Tissue engineered blood vessels

ABSTRACT

The invention is directed to methods for preparing artificial blood vessels by preconditioning a matrix seeded with endothelial cells to fluid flow conditions that mimic blood flow.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/664,212, filed Mar. 21, 2005; and U.S. Provisional Application No.60/660,832, filed Mar. 11, 2005; the contents of both applications beinghereby incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

The technical field of this invention relates to matrices for cellularattachment and growth. The invention also relates to methods of makingand using these matrices for tissue engineering and the construction ofartificial blood vessels.

Diseases of small and medium caliber arteries account for the majorityof deaths in the United States each year. Over 500,000 coronary bypassgrafts and 50,000 peripheral bypass grafts are performed annually inEurope and in the United States. However, up to 30% of the patients whorequire arterial bypass surgery lack suitable or sufficient amounts ofsuitable autologous conduits such as small caliber arteries or saphenousveins, which remain the standard conduit for coronary bypass surgery.

Synthetic grafts, such as polytetrafluoroethylene (PTFE) or Dacron(polyethylene terephthalate fiber) have been used to bypass largecaliber high-flow blood vessels. However, these grafts fail when used tobypass small-caliber, low flow blood vessels due to increasedthrombogenicity and accelerated intimal thickening leading to earlygraft stenosis and occlusion.

In the last two decades many attempts have been made to engineer patentsmall-caliber (<5-6 mm) arterial substitutes. However, these substitutesexhibited poor mechanical and burst strengths which precluded in vivoimplantation.

Accordingly, a need exists for creating improved matrices for tissueengineering of blood vessels. In particular, a need exists for creatingmatrices that have a similar composition and ultrastructure to nativescaffold materials that can locally deliver a therapeutic agent thataids in developing the artificial tissue construct.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions for preparingtissue engineered blood vessels. These blood vessels can be made fromelectrospun matrices or decellularized matrices, seeded with endothelialcells and subjected to a preconditioning under physiological fluidconditions that mimic the blood flow rate and pulse rate of nativevessels. The method involves using a preconditioning chamber, into whichthe seeded matrices are placed. The seeded scaffolds are then subject tovarying fluid flow parameters in which biological fluid flows throughthe seeded vessel in a closed loop system. The method also involvesusing biological fluid that has the consistency of blood and plasma.Continued growth and differentiation of the tissue layer on the matrixunder fluid flow conditions results in the formation of tissueengineered blood vessels, that function as native in vivo blood vessels.The matrices may also be coupled with nanoparticles and therapeuticagents for controlled delivery of therapeutic agents, and/or coupledwith image enhancing agents to monitor the tissue and scaffoldremodeling in vivo.

Accordingly, in one aspect, the invention pertains to a method forproducing a preconditioned blood vessel by providing a biocompatiblematrix shaped in a tubular configuration seeded with a culturedpopulation of endothelial cells on the inside surface of the matrix. Thefirst and second end of the tubular matrix can be attached to a firstand second attachment element in the preconditioning chamber(bioreactor). The first and second attachment elements each having achannel that is fluidly coupled to fluid flow system. To seed theendothelial cells attach to the inside surface of the tubular matrix,the preconditioning chamber can be gently rotated. After the cells haveseeded on the matrix, the seeded tubular matrix can be preconditionedwith the fluid flow system in which a biological fluid is moved throughthe seeded tubular matrix. The flow-rate and pulse-rate of thebiological fluid can controlled such that a preconditioned blood vesselis produced.

The biocompatible matrix can be a decellularized matrix, an electrospunmatrix and a synthetic polymer matrix. In one embodiment, the matrix isa decellularized matrix. The endothelial cells can be isolated forexample from a human saphenous vein, or derived from progenitor cellsisolated from peripheral blood or bone marrow. The preconditioningchamber is a container with dimensions suitable for attaching a lengthof tubular matrix. For example, the shape of a rectangular box withholes at each end to which attachment elements can be added. Theattachment elements hold the ends of the seeded matrix in an openconfiguration such that biological fluid can pass from one end of thepreconditioning chamber through the seeded matrix and out from the otherend of the chamber in a closed loop fluid flow system.

The step of preconditioning the matrix can involve moving the biologicalfluid through the inside surface of the attached matrix in a closedfluid flow system. This allows the inside of the seeded matrix to becomepreconditioned to the fluid flow and allows the seeded cells to developunder fluid flow conditions as they would in the native blood vessel.The step of preconditioning involves moving the biological fluid throughthe inside surface of the attached matrix as a continuous flow, forexample at a flow-rate that can be incremented over time to induce awall shear in the range of about 1 dyne/cm² to about 30 dynes/cm². Thestep of preconditioning the matrix can also involve moving thebiological fluid through the inside surface of the attached matrix as apulsed flow, for example, a pulsed flow that has a pulse-rate which isincremented over time to induce a wall shear in the range of about 10dynes/cm² to about 45 dynes/cm². The pulse-rate can be incremented overtime to induce a wall pressure distribution in the engineered bloodvessel in the range of about 60 to about 200 mmHg.

The biological fluid can be moved through the seeded tubular matrix by apump such as a mechanical pump. The biological fluid is one that canreadily move through the lumen of the seeded matrix. Examples fluidsinclude, but are not limited to, culture medium, buffer medium, andphysiological medium. The composition and viscosity of the biologicalfluid can be altered to be the equivalent to blood. This can beaccomplished by adding high molecular weight proteins such as 40% of 100kDa dextran to the culture medium, buffer medium, and physiologicalmedium.

The step of preconditioning may also involve preconditioning theexterior of the matrix by seeding the exterior matrix with anotherpopulation of cells that can be the same or different from theendothelial cell population, and exposing the exterior seeded matrix toa biological fluid. This can be done by adding a volume of biologicalfluid to the preconditioning chamber such that the outside surface ofthe tubular matrix is exposed to the biological fluid. In oneembodiment, the biological fluid used to precondition the inside of thevessel is the same as the biological fluid used to precondition theoutside of the vessel. In another embodiment, the biological fluid usedto precondition the inside of the vessel is different from thebiological fluid used to precondition the outside of the vessel. Forexample, the inside of the matrix (lumen side) can be seeded withendothelial cells and preconditioned with a biological fluid that isoptimal for endothelial cell growth and proliferation, while the outsideof the matrix can be seeded with smooth muscle cells and preconditionedwith a biological fluid that is optimal for smooth muscle cell growthand proliferation. It will be appreciated that the fluid flow parameterscan be separately controlled to provide the optimum preconditioning forthe inside and the outside of the seeded matrix.

In other embodiments, the matrix can be an electrospun matrix comprisingat least one natural component and at least one synthetic polymercomponent and a therapeutic agent coupled to a nanoparticle. The naturalcomponent can be collagen and the synthetic polymer component canpoly(lactide-co-glycolides) (PLGA). The electrospun matrix may furthercomprise elastin. The therapeutic agent can be heparin and thenanoparticle can be a quantum dot. The heparin and quantum dot can beencapsulated in a polymer and the heparin release from the nanoparticlecan be locally controlled by the application of radiation at awavelength in the range of about 700 nm to about 1000 nm. The heparincan be locally released from the nanoparticle by heating thenanoparticle so that it alters the ultrastructure of the polymer torelease the heparin. In other embodiments, the nanospun matrix canfurther comprise an image enhancing agent such as gadolinium.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of an electrospin apparatus;

FIG. 2 is a schematic of the conjugation of heparin on quantum dots;

FIG. 3 is an electrospin nanofiber;

FIG. 4 is graph of pressure-diameter curves of vascular graft scaffolds;

FIG. 5A is a graph of axial and circumferential stress-strain data fromuniaxial testing of two decellularized constructs;

FIG. 5B is a graph of axial and circumferential stress-strain data fromuniaxial testing of an electrospun vessel;

FIG. 6A is a graph of cell viability of endothelial cells cultured onfour matrices;

FIG. 6B is a graph of mitochondrial metabolic activity of endothelialcells cultured on four matrices;

FIG. 7A is a graph of cell viability of endothelial cells cultured onfive matrices;

FIG. 7B is a graph of mitochondrial metabolic activity of endothelialcells cultured on five matrices;

FIG. 8A is a graph of optical intensity versus time for a salinesuspension having heparin-containing capsules before luminescence andten minutes after luminescence;

FIG. 8B is a graph of heparin release by microcapsules upon nearinfra-red irradiation;

FIG. 9 is a graph showing the quantification of remaining heparin fromretrieved vascular scaffolds;

FIG. 10A is a graph showing the pressure-diameter curve ofdecellularized and native blood vessels;

FIG. 10B is a graph showing the axial and circumferential stress andstrain of decellularized and native blood vessels;

FIG. 11A is a schematic top view of a preconditioning chamber of theinvention;

FIG. 11B is a schematic side view of a preconditioning chamber of theinvention;

FIG. 11C is a schematic traverse view of a preconditioning chamber ofthe invention;

FIG. 12 is a photograph of a preconditioning chamber of the invention;

FIG. 13 is a graph showing the flow rate waveform of a pulsatile flowwithin a preconditioning chamber of the invention;

FIG. 14A is a bar graph showing the production of 6-ket-PGF1 fromdecellularized blood vessels seeded with human endothelial cells; and

FIG. 14B is a graph showing the production of nitric oxide fromdecellularized blood vessels seeded with human endothelial cells.

DETAILED DESCRIPTION

So that the invention may more readily be understood, certain terms arefirst defined:

The term “attach” or “attaches” as used herein refers to cells thatadhere directly or indirectly to a substrate as well as to cells thatadhere to other cells.

The phrase “biocompatible substrate” as used herein refers to a materialthat is suitable for implantation into a subject onto which a cellpopulation can be deposited. A biocompatible substrate does not causetoxic or injurious effects once implanted in the subject. In oneembodiment, the biocompatible substrate is a polymer with a surface thatcan be shaped into the desired structure that requires repairing orreplacing. The polymer can also be shaped into a part of an structurethat requires repairing or replacing. The biocompatible substrateprovides the supportive framework that allows cells to attach to it, andgrow on it. Cultured populations of cells can then be grown on thebiocompatible substrate, which provides the appropriate interstitialdistances required for cell-cell interaction.

The term “subject” as used herein is intended to include livingorganisms in which an immune response is elicited. Preferred subjectsare mammals. Examples of subjects include but are not limited to,humans, monkeys, dogs, cats, mice, rates, cows, horses, pigs, goats andsheep.

The term “decellularized” or “decellularization” as used herein refersto a biostructure (e.g., an organ, or part of an organ), from which thecellular and tissue content has been removed leaving behind an intactacellular infra-structure. Organs such as the kidney are composed ofvarious specialized tissues. The specialized tissue structures of anorgan, or parenchyma, provide the specific function associated with theorgan. The supporting fibrous network of the organ is the stroma. Mostorgans have a stromal framework composed of unspecialized connectingtissue which supports the specialized tissue. The process ofdecellularization removes the specialized tissue, leaving behind thecomplex three-dimensional network of connective tissue. The connectivetissue infra-structure is primarily composed of collagen. Thedecellularized structure provides a biocompatible substrate onto whichdifferent cell populations can be infused. Decellularized biostructurescan be rigid, or semi-rigid, having an ability to alter their shapes.Examples of decellularized organs useful in the present inventioninclude, but are not limited to, the heart, kidney, liver, pancreas,spleen, bladder, ureter and urethra.

The phrase “three-dimensional scaffold” as used herein refers to theresidual infra-structure formed when a natural biostructure, e.g. anorgan, is decellularized. This complex, three-dimensional, scaffoldprovides the supportive framework that allows cells to attach to it, andgrow on it. Cultured populations of cells can then be grown on thethree-dimensional scaffold, which provides the exact interstitialdistances required for cell-cell interaction. This provides areconstructed organ that resembles the native in vivo organ. Thisthree-dimensional scaffold can be perfused with a population of culturedcells, e.g., endothelial cells, which grow and develop to provide anendothelial tissue layer capable of supporting growth and development ofat least one additional cultured cell population.

The term “natural biostructure” as used herein refers to a biologicalarrangement found within a subject, for example, organs, that includebut are not limited, heart, kidney, liver, pancreas, spleen, bladder,ureter and urethra. The term “natural biostructure” is also intended toinclude parts of biostructures, for example parts of organs, forexample, the renal artery of a kidney.

The terms “electrospinning” or “electrospun,” as used herein refers toany method where materials are streamed, sprayed, sputtered, dripped, orotherwise transported in the presence of an electric field. Theelectrospun material can be deposited from the direction of a chargedcontainer towards a grounded target, or from a grounded container in thedirection of a charged target. In particular, the term “electrospinning”means a process in which fibers are formed from a charged solutioncomprising at least one natural biological material, at least onesynthetic polymer material, or a combination thereof by streaming theelectrically charged solution through an opening or orifice towards agrounded target.

A natural biological material can be a naturally occurring organicmaterial including any material naturally found in the body of a mammal,plant, or other organism. A synthetic polymer material can be anymaterial prepared through a method of artificial synthesis, processing,or manufacture. Preferably the synthetic materials is a biologicallycompatible material. The natural or synthetic materials are also thosethat are capable of being charged under an electric field.

The terms “solution” and “fluid” is used in the context of producing anelectrospun matrix and describes a liquid that is capable of beingcharged and which comprises at least one natural material, at least onesynthetic material, or a combination thereof. In a preferred embodiment,the fluid comprises at least one type of collagen, an additional naturalmaterial such as at least one type of elastin and at least one syntheticpolymer, e.g., poly-L glycolic acid (PLGA).

The term “co-polymer” as used herein is intended to encompassco-polymers, ter-polymers, and higher order multiple polymercompositions formed by block, graph or random combination of polymericcomponents.

The terms “nanoparticles,” “nanostructures,” and “quantum dots” are usedinterchangeably herein to describe materials having dimensions of theorder of one or a few nanometers to a few micrometers, more preferablyfrom about 1 to about 1000 nanometers.

The term “preconditioning chamber” as used herein refers to a containerthat allows a matrix seeded with cells to be conditioned such that thecells on the matrix develop under physiological conditions. For example,to create blood vessels, a matrix can be seeded with endothelial cellsand the endothelial cells allowed to develop under native fluidconditions such as pulsed conditions that mimic the pulse rate of bloodthrough native vessels, or fluid flow conditions with alterations inpressure. To begin with, the pulse rate and the flow rate can be slowuntil the cells adjust to this pulse-rate or flow-rate, the flow-rateand pulse-rate can then gradually be increased until the cells adjust tothe new pulse-rate and flow-rate an so forth. By gradually increasingthe pulse-rate and the flow-rate, the vessels become conditioned tobeing able to withstand pressure as high as those produced during eachheartbeat.

The biological fluid can be moved through the inside surface of theattached matrix (lumen) as a continuous flow, for example at a flow-ratethat can be incremented over time to induce a wall shear in the range ofabout 1 dyne/cm² to about 30 dynes/cm². The step of preconditioning thematrix can also involve moving the biological fluid through the insidesurface of the attached matrix as a pulsed flow, for example, a pulsedflow that has a pulse-rate which is incremented over time to induce awall shear in the range of about 10 dynes/cm² to about 45 dynes/cm². Thepulse-rate can be incremented over time to induce a wall pressuredistribution in the engineered blood vessel in the range of about 60 toabout 200 mmHg. A different of the same biological fluid can also beused to precondition the outside of the matrix.

The term “biological fluid” as used herein refers a liquid that can beused to precondition an engineered blood vessel. The biological fluidhas a composition and viscosity that mimics blood so that the engineeredblood vessels are exposed to the same fluid flow dynamics as nativeblood vessels. Examples of biological fluids can include any buffer,medium of physiological fluid (e.g., DMEM with 10% FCS with a bloodviscosity). The viscosity of the fluids can be altered by adding highmolecular weight proteins such as 100 kDa dextran. Other molecularweight dextrans can also be used. It will be appreciated that the amountof dextran to be used depends on the molecular weight and can range fromabout 10%, 20%, 30%, 40%, 50%, and 60%. The composition may also bevaried by adding other blood like constituents such as salts.

I Electrospun Matrices

The invention pertains to methods and compositions for producing andusing electrospun matrices. The process of electrospinning generallyinvolves the creation of an electrical field at the surface of a liquid.The resulting electrical forces create a jet of liquid which carrieselectrical charge. The liquid jets may be attracted to otherelectrically charged objects at a suitable electrical potential. As thejet of liquid elongates and travels, it will harden and dry. Thehardening and drying of the elongated jet of liquid may be caused bycooling of the liquid, i.e., where the liquid is normally a solid atroom temperature; evaporation of a solvent, e.g., by dehydration,(physically induced hardening); or by a curing mechanism (chemicallyinduced hardening). The produced fibers are collected on a suitablylocated, oppositely charged target substrate.

The electrospinning apparatus includes an electrodepositing mechanismand a target substrate. The electrodepositing mechanism includes atleast one container to hold the solution that is to be electrospun. Thecontainer has at least one orifice or nozzle to allow the streaming ofthe solution from the container. If there are multiple containers, aplurality of nozzles may be used. One or more pumps (e.g., a syringepump) used in connection with the container can be used to control theflow of solution streaming from the container through the nozzle. Thepump can be programmed to increase or decrease the flow at differentpoints during electrospinning.

The electrospinning occurs due to the presence of a charge in either theorifices or the target, while the other is grounded. In someembodiments, the nozzle or orifice is charged and the target isgrounded. Those of skill in the electrospinning arts will recognize thatthe nozzle and solution can be grounded and the target can beelectrically charged.

The target can also be specifically charged or grounded along apreselected pattern so that the solution streamed from the orifice isdirected into specific directions. The electric field can be controlledby a microprocessor to create an electrospun matrix having a desiredgeometry. The target and the nozzle or nozzles can be engineered to bemovable with respect to each other thereby allowing additional controlover the geometry of the electrospun matrix to be formed. The entireprocess can be controlled by a microprocessor that is programmed withspecific parameters that will obtain a specific preselected electrospunmatrix.

In embodiments in which two materials combine to form a third material,the solutions containing these components can be mixed togetherimmediately before they are streamed from an orifice in theelectrospinning procedure. In this way, the third material formsliterally as the microfibers in the electrospinning process.

While the following is a description of a preferred method, otherprotocols can be followed to achieve the same result. In FIG. 1, acontainer 10, (e.g., a syringe or micropipette), with an orifice ornozzle 12 (e.g., a Taylor cone), is filled with a solution with at leastone natural material, and at least one synthetic material 14. Thecontainer 10 is suspended opposite a grounded target 16, such as a metalground screen. A fine wire 18 is placed in the solution to charge thesolution in the container to a high voltage. At a specific voltagedetermined for each solution, the solution in the container nozzle isdirected towards the grounded target. The single jet stream 20 ofmaterials forms a splayed jet 22, upon reaching the grounded target,e.g., a rapidly rotating mandrel. The splayed jet collects and dries toform a three-dimensional, ultra thin, interconnected matrix ofelectrospun fibers. In some embodiments, a plurality of containers canbe used with each of the containers holding a different compound.

Minimal electrical current is involved in the electrospinning process,therefore the process does not denature the materials that form theelectrospun matrix, because the current causes little or no temperatureincrease in the solutions during the procedure.

The electrospinning process can be manipulated to meet the specificrequirements for any given application of the electrospun matrix. In oneembodiment, a syringe can be mounted on a frame that moves in the x, yand z planes with respect to the grounded substrate. In anotherembodiment, a syringe can be mounted around a grounded substrate, forinstance a tubular mandrel. In this way, the materials that form thematrix streamed from the a syringe can be specifically aimed orpatterned. Although the micropipette can be moved manually, the frameonto which the a syringe is mounted can also be controlled by amicroprocessor and a motor that allows the pattern of streaming to bepredetermined. Such microprocessors and motors are known to one ofordinary skill in the art, for example matrix fibers can be oriented ina specific direction, they can be layered, or they can be programmed tobe completely random and not oriented.

The degree of branching can be varied by many factors including, but notlimited to, voltage (for example ranging from about 0 to 30,000 volts),distance from a syringe tip to the substrate (for example from 1-100 cm,0-40 cm, and 1-10 cm), the speed of rotation, the shape of the mandrel,the relative position of the a syringe tip and target (i.e. in front of,above, below, aside etc.), and the diameter of a syringe tip(approximately 0-2 mm), and the concentration and ratios of compoundsthat form the electrospun matrix. Other parameters which are importantinclude those affecting evaporation of solvents such as temperature,pressure, humidity. The molecular weight of the polymer improves itsability to entangle and form fibers, and polymers with the molecularweight of 100 kDa generally performed. Those skilled in the art willrecognize that these and other parameters can be varied to formelectrospun materials with characteristics that are particularly adaptedfor specific applications.

The geometry of the grounded target can be modified to produce a desiredmatrix. By varying the ground geometry, for instance having a planar orlinear or multiple points ground, the direction of the streamingmaterials can be varied and customized to a particular application. Forinstance, a grounded target comprising a series of parallel lines can beused to orient electrospun materials in a specific direction. Thegrounded target can be a cylindrical mandrel whereby a tubular matrix isformed. The ground can be variable surface that can be controlled by amicroprocessor that dictates a specific ground geometry that isprogrammed into it. Alternatively, the ground can be mounted on a framethat moves in the x, y, and z planes with respect to a stationarycontainer, e.g., a syringe or micropipette tip.

Electrospinning allows great flexibility and allows for customizing theconstruct to virtually any shape needed. In shaping matrices, portionsof the matrix may be sealed to one another by, for example, heatsealing, chemical sealing, and application of mechanical pressure or acombination thereof. The electrospun compositions may be shaped intoshapes such as a skin patch, an intraperitoneal implant, a subdermalimplant, the interior lining of a stent, a cardiovascular valve, atendon, a ligament, a muscle implant, a nerve guide and the like.

The electrospinning process can also be modified for example by (i)using mixed solutions (for example, materials along with substances suchas cells, growth factors, or both) in the same container to producefibers composed of electrospun compounds as well as one or moresubstances to produce a “blend,” and (ii) applying agents such as Teflononto the target to facilitate the removal of electrospun compounds fromthe target (i.e., make the matrix more slippery so that the electrospunmatrix does not stick to the target).

The various properties of the electrospun materials can be adjusted inaccordance with the needs and specifications of the cells to besuspended and grown within them. The porosity, for instance, can bevaried in accordance with the method of making the electrospun materialsor matrix. Electrospinning a particular matrix, for instance, can bevaried by fiber size and density. If the cells to be grown in the matrixrequire a high nutrient flow and waste expulsion, then a loose matrixcan be created. On the other hand, if the tissue to be made requires adense environment, then a dense matrix can be designed. Porosity can bemanipulated by mixing salts or other extractable agents. Removing thesalt will leave holes of defined sizes in the matrix.

One embodiment for appropriate conditions for electrospinning a matrixis as follows. For electrospinning a matrix by combining 45% collagen I,15% elastin and 40% PLGA, the appropriate approximate ranges are:voltage 0-30,000 volts (10-100 kV potential preferably 15-30 kV); pH 7.0to 8.0; temperature 20 to 40° C., e.g., room temperature of 25° C.; andthe distance from the container to the grounded plate can range fromabout 1 cm to about 100 cm, preferably about 1 cm to 10 cm. In additionto depositing the charged fibers on the grounded plate, the fibers canbe deposited onto another substrate such as a stainless steel mandrel.The mandrel can be rotated at 20-1000 rpm, preferably about 300-700 rpm.

Examples of naturally occurring materials include, but are not limitedto, amino acids, peptides, denatured peptides such as gelatin fromdenatured collagen, polypeptides, proteins, carbohydrates, lipids,nucleic acids, glycoproteins, lipoproteins, glycolipids,glycosaminoglycans, and proteoglycans. In a preferred embodiment, thematerials compound is an extracellular matrix material, including butnot limited to collagen, fibrin, elastin, laminin, fibronectin,hyaluronic acid, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatansulfate, heparin sulfate, heparin, and keratan sulfate, andproteoglycans. These materials may be isolated from humans or otheranimals or cells. A preferred natural compound is collagen. Examples ofcollagen include, but are not limited to collagen I, collagen II,collagen III, collagen IV, collagen V, collagen VI, collagen VII,collagen VIII, collagen IX, and collagen X. Another preferred naturalcompound is elastin. Elastin fibers are responsible for the elasticproperties of several tissues. Elastin is found, for example, in skin,blood vessels, and tissues of the lung where it imparts strength,elasticity and flexibility.

One class of synthetic polymer materials are biocompatible syntheticpolymers. Such polymers include, but are not limited to,poly(urethanes), poly(siloxanes) or silicones, poly(ethylene),poly(vinyl pyrrolidone), poly(2-hydroxy ethyl methacrylate),poly(N-vinyl pyrrolidone), poly(methyl methacrylate), poly(vinylalcohol) (PVA), poly(acrylic acid), poly(vinyl acetate), polyacrylamide,poly(ethylene-co-vinyl acetate), poly(ethylene glycol), poly(methacrylicacid), polylactic acid (PLA), polyglycolic acids (PGA),poly(lactide-co-glycolides) (PLGA), nylons, polyamides, polyanhydrides,poly(ethylene-co-vinyl alcohol) (EVOH), polycaprolactone, poly(vinylacetate), polyvinylhydroxide, poly(ethylene oxide) (PEO) andpolyorthoesters or any other similar synthetic polymers that may bedeveloped that are biologically compatible. A preferred syntheticpolymer is PGLA.

In matrices composed of electrospun elastin (for elasticity),electrospun collagen (to promote cell infiltration and lend mechanicalintegrity), and other components, such as PLGA, PGA, PLA, PEO, PVA, orother blends, the relative ratio of the different components in thematrix can be tailored to specific applications (e.g. more elastin, lesscollagen depending on the tissue to be engineered).

Electrospun matrices can be formed of electrospun fibers of syntheticpolymers that are biologically compatible. The term “biologicallycompatible” includes copolymers and blends, and any other combinationsof the forgoing either together or with other polymers. The use of thesepolymers will depend on given applications and specifications required.A more detailed discussion of these polymers and types of polymers isset forth in Brannon-Peppas, Lisa, “Polymers in Controlled DrugDelivery,” Medical Plastics and Biomaterials, November 1997, which isincorporated herein by reference.

When both natural and synthetic materials are used in an electrospunmatrix, the natural material component can range from about 5 percent toabout 95 percent, preferably from about 25 percent to about 75 percentby weight. The synthetic material component can range from about 5percent to about 95 percent, preferably from about 25 percent to about75 percent by weight. In certain embodiments, both collagen and elastincan be included as natural material components, preferably with apredominance of collagen, e.g., greater than 40 percent of the naturalmaterial component. Ratios of collagen, elastin, and PLGA may betailored to fit the application: for instances, normal levels ofcollagen and elastin vary from the more elastic vessels closer to theheart to less compliant vessels further from the heart. A vessel such asthe aorta would have greater elastin content than a distal vessel. Thepercentages of collagen I, elastin, and other collagens (collagen IIIfor blood vessels or collagen II, for instance, for cartilage) may bewhatever is desired, as long as the molecular weight of these collagensis sufficient to form fibers in the electrospinning process. Ratios ofcollagen I may be from 40% to 80%, or 50% to 100%. Elastin may also beused in higher ratios from 5% to 50%. PLGA or another syntheticbiodegradable polymer may be used as desired in ratios from 5 to 80%.For a completely biological substrate, synthetic polymers may be omittedcompletely and only biological polymers may be used.

The compounds to be electrospun can be present in the solution at anyconcentration that will allow electrospinning. In one embodiment, thecompounds may be electrospun are present in the solution atconcentrations between 0 and about 1.000 g/ml. In another embodiment,the compounds to be electrospun are present in the solution at totalsolution concentrations between 10-15 w/v % (100-150 mg/ml or 0-0.1g/L).

The compounds can be dissolved in any solvent that allows delivery ofthe compound to the orifice, tip of a syringe, under conditions that thecompound is electrospun. Solvents useful for dissolving or suspending amaterial or a substance will depend on the compound. Electrospinningtechniques often require more specific solvent conditions. For example,collagen can be electrodeposited as a solution or suspension in water,2,2,2-trifluoroethanol, 1,1,1,3,3,3-hexafluoro-2-propanol (also known ashexafluoroisopropanol or HFIP), or combinations thereof. Fibrin monomercan be electrodeposited or electrospun from solvents such as urea,monochloroacetic acid, water, 2,2,2-trifluoroethanol, HFIP, orcombinations thereof. Elastin can be electrodeposited as a solution orsuspension in water, 2,2,2-trifluoroethanol, isopropanol, HFIP, orcombinations thereof, such as isopropanol and water. In one desirableembodiment, elastin is electrospun from a solution of 70% isopropanoland 30% water containing 250 mg/ml of elastin. Other lower orderalcohols, especially halogenated alcohols, may be used. Other solventsthat may be used or combined with other solvents in electrospinningnatural matrix materials include acetamide, N-methylformamide,N,N-dimethylformamide (DMF), dimethylsulfoxide (DMSO),dimethylacetamide, N-methyl pyrrolidone (NMP), acetic acid,trifluoroacetic acid, ethyl acetate, acetonitrile, trifluoroaceticanhydride, 1,1,1-trifluoroacetone, maleic acid, hexafluoroacetone.Organic solvents such as methanol, chloroform, and trifluoroethanol(TFE) and emulsifying agents.

The selection of a solvent is based in part on consideration ofsecondary forces that stabilize polymer-polymer interactions and thesolvent's ability to replace these with strong polymer-solventinteractions. In the case of polypeptides such as collagen, and in theabsence of covalent crosslinking, the principal secondary forces betweenchains are: (1) coulombic, resulting from attraction of fixed charges onthe backbone and dictated by the primary structure (e.g., lysine andarginine residues will be positively charged at physiological pH, whileaspartic or glutamic acid residues will be negatively charged); (2)dipole-dipole, resulting from interactions of permanent dipoles; thehydrogen bond, commonly found in polypeptides, is the strongest of suchinteractions; and (3) hydrophobic interactions, resulting fromassociation of non-polar regions of the polypeptide due to a lowtendency of non-polar species to interact favorably with polar watermolecules. Therefore, solvents or solvent combinations that canfavorably compete for these interactions can dissolve or dispersepolypeptides. For example, HFIP and TFE possess a highly polar OH bondadjacent to a very hydrophobic fluorinated region. While not wanting tobe bound by the following theories, it is believed that the alcoholportion can hydrogen bond with peptides, and can also solvate charges onthe backbone, thus reducing Coulombic interactions between molecules.Additionally, the hydrophobic portions of these solvents can interactwith hydrophobic domains in polypeptides, helping to resist the tendencyof the latter to aggregate via hydrophobic interactions. It is furtherbelieved that solvents such as HFIP and TFE, due to their lower overallpolarities compared to water, do not compete well for intramolecularhydrogen bonds that stabilize secondary structures such as an alphahelix. Consequently, alpha helices in these solvents are believed to bestabilized by virtue of stronger intramolecular hydrogen bonds. Thestabilization of polypeptide secondary structures in these solvents isbelieved desirable, especially in the cases of collagen and elastin, topreserve the proper formation of collagen fibrils duringelectrospinning.

In one embodiment, the solvent has a relatively high vapor pressure topromote the stabilization of an electrospinning jet to create a fiber asthe solvent evaporates. In embodiments involving higher boiling pointsolvents, it is often desirable to facilitate solvent evaporation bywarming the spinning or spraying solution, and optionally theelectrospinning stream itself, or by electrospinning in reducedatmospheric pressure. It is also believed that creation of a stable jetresulting in a fiber is facilitated by a high surface tension of thepolymer/solvent mixture.

Similar to conventional electrospinning, midair electrospinning can beused which employs the same experimental set-up as other electrospinningtechniques. However, in order to precipitate fibers before they reachthe grounded target, the distance from the needle to the grounded targetcan be increased. For example, increasing the distance from the 10-30 cmto a distance of 30-40 cm. The field strength can be maintained oraltered by increasing the applied potential at the needle tip.Increasing the distance from the needle tip to the grounded targetallows the polymer jet to experience a longer “flight time.” The addedflight time, allows the solvent to be completely evaporated from the jetallowing the fibers to fully develop.

By varying the composition of the fibers being electrospun, it will beappreciated that fibers having different physical or chemical propertiesmay be obtained. This can be accomplished either by spinning a liquidcontaining a plurality of components, each of which may contribute adesired characteristic to the finished product, or by simultaneouslyspinning fibers of different compositions from multiple liquid sources,that are then simultaneously deposited to form a matrix. The resultingmatrix comprises layers of intermingled fibers of different compounds.This plurality of layers of different materials can convey a desiredcharacteristic to the resulting composite matrix with each differentlayer providing a different property, for example one layer maycontribute to elasticity while another layer contributes to themechanical strength of the composite matrix. These methods can be usedto create tissues with multiple layers such as blood vessels.

The electrospun matrix has an ultrastructure with a three-dimensionalnetwork that supports cell growth, proliferation, differentiation anddevelopment. The spatial distance between the fibers plays an importantrole in cells being able to obtain nutrients for growth as well as forallowing cell-cell interactions to occur. Thus, in various embodimentsof the invention, the distance between the fibers may be about 50nanometers, about 100 nanometers, about 150 nanometers, about 200nanometers, about 250 nanometers, about 300 nanometers, about 350nanometers, about 600 nanometers, about 750 nanometers, about 800nanometers, about 850 nanometers, about 900 nanometers, about 950nanometers, about 1000 nanometers (1 micron), 10 microns, 10 microns, 50microns, about 100 microns, about 150 microns, about 200 microns, about250 microns, about 300 microns, about 350 microns, about 400 microns,about 450 microns, or about 500 microns. In various embodiments thedistance between the fibers may be less than 50 nanometers or greaterthan 500 microns and any length between the quoted ranges as well asintegers.

Additionally, in various embodiments of the invention, the fibers canhave a diameter of about 50 nanometers, about 100 nanometers, about 150nanometers, about 200 nanometers, about 250 nanometers, about 300nanometers, about 350 nanometers, about 600 nanometers, about 750nanometers, about 800 nanometers, about 850 nanometers, about 900nanometers, about 950 nanometers, about 1000 nanometers (1 micron), 50microns, about 100 microns, about 150 microns, about 200 microns, about250 microns, about 300 microns, about 350 microns, about 400 microns,about 450 microns, or about 500 microns, or the diameter may be lessthan 50 nanometers or greater than 500 microns and any diameter betweenthe quoted ranges as well as integers.

The pore size in an electrospun matrix can also be controlled throughmanipulation of the composition of the material and the parameters ofelectrospinning. In some embodiments, the electrospun matrix has a poresize that is small enough to be impermeable to one or more types ofcells. In one embodiment, the average pore diameter is about 500nanometers or less. In another embodiment, the average pore diameter isabout 1 micron or less. In another embodiment, the average pore diameteris about 2 microns or less. In another embodiment, the average porediameter is about 5 microns or less. In another embodiment, the averagepore diameter is about 8 microns or less. Some embodiments have poresizes that do not impede cell infiltration. In another embodiment, thematrix has a pore size between about 0.1 and about 100 μm². In anotherembodiment, the matrix has a pore size between about 0.1 and about 50μm². In another embodiment, the matrix has a pore size between about 1.0μm and about 25 μm. In another embodiment, the matrix has a pore sizebetween about 1.0 μm and about 5 μm. Infiltration can also beaccomplished with implants with smaller pore sizes. The pore size of anelectrospun matrix can be readily manipulated through control of processparameters, for example by controlling fiber deposition rate throughelectric field strength and mandrel motion, by varying solutionconcentration (and thus fiber size). Porosity can also be manipulated bymixing porogenic materials, such as salts or other extractable agents,the dissolution of which will leave holes of defined sizes in thematrix. The pore size can also be controlled by the amount ofcross-linking present in the matrix.

The mechanical properties of the matrix will depend on the polymermolecular weight and polymer type/mixture. It will also depend onorientation of the fibers (preferential orientation can be obtained bychanging speed of a rotating or translating surface during the fibercollection process), fiber diameter and entanglement. The crosslinkingof the polymer will also effect its mechanical strength after thefabrication process.

The electrospun matrix can be cross linked to increase its stability andstrength. The crosslinking can generally be conducted at roomtemperature and neutral pH conditions, however, the conditions may bevaried to optimize the particular application and crosslinking chemistryutilized. For crosslinking using the EDC chemistry with NHS in MES/EtOH,pH of from 4.0 to 8.0 and temperatures from 0° C. to room temperature(25° C.) for two hours, can be used. It is known that highertemperatures are unpreferred for this chemistry due to decomposition ofEDC. Similarly, basic pH (e.g., 8-14) is also unpreferred for thisreason when using this chemistry. Other crosslinking chemistries canalso be used for example, by soaking the electrospun matrix in 20%dextran solution (to reduce shrinking), followed by 1% glutaraldehydesolution. Yet other cross-linking chemistries involve usingpoly(ethylene glycol) (PEG) as a spacer in a crosslinking agent with anN-protected amino acid.

II. Decellularized Matrices

Natural biostructures, e.g. a blood vessel or an organ, can be obtainedfrom a donor of the same species as the subject, for example, a humancadaver blood vessel or organ for a human recipient. The naturalbiostructure can also be obtained from a different species whichincludes, but is not limited to, monkeys, dogs, cats, mice, rats, cows,horses, pigs, goats and sheep. The natural biostructure can also beobtained from the subject requiring a reconstructed organ and can havethe dysfunctional blood vessel removed and decellularized using theprocess described below. The decellularized blood vessel of the subjectcan be used as the three-dimensional scaffold to reconstruct anartificial blood vessel using cultured endothelial cells (e.g., humanendothelial cells) isolated from the subject. In one embodiment, theendothelial cells are isolated from the human saphenous vein. In anotherembodiment, the endothelial cells can be produced from progenitor cellsisolated from the blood or bone marrow of the subject. The artificialreconstructed blood vessel can be implanted back into the subject forfurther development.

Biostructures, e.g., blood vessels, or parts of blood vessels can bedecellularized by removing the entire cellular and tissue content fromthe blood vessel. The decellularization process comprises a series ofsequential extractions. One key feature of this extraction process isthat harsh extraction that may disturb or destroy the complexinfra-structure of the biostructure, be avoided. The first step involvesremoval of cellular debris and solubilization of the cell membrane. Thisis followed by solubilization of the nuclear cytoplasmic components anthe nuclear components.

Preferably, the biostructure, e.g., an a blood vessel, is decellularizedby removing the cell membrane and cellular debris surrounding the bloodvessel using gentle mechanical disruption methods. The gentle mechanicaldisruption methods must be sufficient to disrupt the cellular membrane.However, the process of decellularization should avoid damage ordisturbance of the biostructure's complex infra-structure. Gentlemechanical disruption methods include scraping the surface of the bloodvessel, agitating the blood vessel, or stirring the blood vessel in asuitable volume of fluid, e.g., distilled water. In one embodiment, thegentle mechanical disruption method includes magnetically stirring(e.g., using a magnetic stir bar and a magnetic plate) the blood in asuitable volume of distilled water until the cell membrane is disruptedand the cellular debris has been removed from the blood vessel.

After the cell membrane has been removed, the nuclear and cytoplasmiccomponents of the biostructure are removed. This can be performed bysolubilizing the cellular and nuclear components without disrupting theinfra-structure. To solubilize the nuclear components, non-ionicdetergents or surfactants may be used. Examples of non-ionic detergentsor surfactants include, but are not limited to, the Triton series,available from Rohm and Haas of Philadelphia, Pa., which includes TritonX-100, Triton N-101, Triton X-114, Triton X-405, Triton X-705, andTriton DF-16, available commercially from many vendors; the Tweenseries, such as monolaurate (Tween 20), monopalmitate (Tween 40),monooleate (Tween 80), and polyoxethylene-23-lauryl ether (Brij. 35),polyoxyethylene ether W-1 (Polyox), and the like, sodium cholate,deoxycholates, CHAPS, saponin, n-Decyl β-D-glucopuranoside, n-heptyl β-Dglucopyranoside, n-Octyl-α-D-glucopyranoside and Nonidet P-40.

One skilled in the art will appreciate that a description of compoundsbelonging to the foregoing classifications, and vendors may becommercially obtained and may be found in “Chemical Classification,Emulsifiers and Detergents”, McCutcheon's, Emulsifiers and Detergents,1986, North American and International Editions, McCutcheon Division, MCPublishing Co., Glen Rock, N.J., U.S.A. and Judith Neugebauer, A Guideto the Properties and Uses of Detergents in Biology and Biochemistry,Calbiochem, Hoechst Celanese Corp., 1987. In one preferred embodiment,the non-ionic surfactant is the Triton series, preferably, Triton X-100.

The concentration of the non-ionic detergent may be altered depending onthe type of biostructure being decellularized. For example, for delicatetissues, e.g., blood vessels, the concentration of the detergent shouldbe decreased. Preferred concentrations ranges non-ionic detergent can befrom about 0.001 to about 2.0% (w/v). More preferably, about 0.05 toabout 1.0% (w/v). Even more preferably, about, 0.1% (w/v) to about 0.8%(w/v). Preferred concentrations of these range from about 0.001 to about0.2% (w/v), with about 0.05 to about 0.1% (w/v) particular preferred.

The cytoskeletal component, comprising consisting of the densecytoplasmic filament networks, intercellular complexes and apicalmicrocellular structures, may be solubilized using alkaline solution,such as, ammonium hydroxide. Other alkaline solution consisting ofammonium salts or their derivatives may also be used to solubilize thecytoskeletal components. Examples of other suitable ammonium solutionsinclude ammonium sulphate, ammonium acetate and ammonium hydroxide. In apreferred embodiment, ammonium hydroxide is used.

The concentration of the alkaline solutions, e.g., ammonium hydroxide,may be altered depending on the type of biostructure beingdecellularized. For example, for delicate tissues, e.g., blood vessels,the concentration of the detergent should be decreased. Preferredconcentrations ranges can be from about 0.001 to about 2.0% (w/v). Morepreferably, about 0.005 to about 0.1% (w/v). Even more preferably,about, 0.01% (w/v) to about 0.08% (w/v).

The decellularized, lyophilized structure may be stored at a suitabletemperature until required for use. Prior to use, the decellularizedstructure can be equilibrated in suitable isotonic buffer or cellculture medium. Suitable buffers include, but are not limited to,phosphate buffered saline (PBS), saline, MOPS, HEPES, Hank's BalancedSalt Solution, and the like. Suitable cell culture medium includes, butis not limited to, RPMI 1640, Fisher's, Iscove's, McCoy's, Dulbecco'smedium, and the like.

III. Synthetic Matrices

The invention also pertains to generating artificial tissue constructsby seeding cultured tissue cells onto or into available biocompatiblematrices. Biocompatible refers to materials that do not have toxic orinjurious effects on biological functions. Biodegradable refers tomaterial that can be absorbed or degraded in a patient's body.Representative materials for forming the biodegradable material includenatural or synthetic polymers, such as, collagen, poly(alpha esters)such as poly(lactate acid), poly(glycolic acid), polyorthoesters amdpolyanhydrides and their copolymers, which degraded by hydrolysis at acontrolled rate and are reabsorbed. These materials provide the maximumcontrol of degradability, manageability, size and configuration.Preferred biodegradable polymer materials include polyglycolic acid andpolyglactin, developed as absorbable synthetic suture material.

Polyglycolic acid and polyglactin fibers may be used as supplied by themanufacturer. Other biodegradable materials include, but are not limitedto, cellulose ether, cellulose, cellulosic ester, fluorinatedpolyethylene, phenolic, poly-4-methylpentene, polyacrylonitrile,polyamide, polyamideimide, polyacrylate, polybenzoxazole, polycarbonate,polycyanoarylether, polyester, polyestercarbonate, polyether,polyetheretherketone, polyetherimide, polyetherketone, polyethersulfone,polyethylene, polyfluoroolefin, polyimide, polyolefin, polyoxadiazole,polyphenylene oxide, polyphenylene sulfide, polypropylene, polystyrene,polysulfide, polysulfone, polytetrafluoroethylene, polythioether,polytriazole, polyurethane, polyvinyl, polyvinylidene fluoride,regenerated cellulose, silicone, urea-formaldehyde, or copolymers orphysical blends of these materials. The material may be impregnated withsuitable antimicrobial agents and may be colored by a color additive toimprove visibility and to aid in surgical procedures.

In some embodiments, attachment of the cells to the biocompatiblesubstrate is enhanced by coating the matrix with compounds such asbasement membrane components, agar, agarose, gelatin, gum arabic,collagens, fibronectin, laminin, glycosaminoglycans, mixtures thereof,and other materials having properties similar to biological matrixmolecules known to those skilled in the art of cell culture. Mechanicaland biochemical parameters ensure the matrix provide adequate supportfor the cells with subsequent growth and proliferation. Factors,including nutrients, growth factors, inducers of differentiation ordedifferentiation, products of secretion, immunomodulators, inhibitorsof inflammation, regression factors, biologically active compounds whichenhance or allow ingrowth of the lymphatic network or nerve fibers, anddrugs, can be incorporated into the matrix or provided in conjunctionwith the matrix. Similarly, polymers containing peptides such as theattachment peptide RGD (Arg-Gly-Asp) can be synthesized for use informing matrices.

Coating refers to coating or permeating a matrix with a material suchas, liquefied copolymers (poly-DL-lactide co-glycolide 50:50 80 mg/mlmethylene chloride) to alter its mechanical properties. Coating may beperformed in one layer, or multiple layers until the desired mechanicalproperties are achieved. These shaping techniques may be employed incombination, for example, a polymeric matrix can be weaved, compressionmolded and glued together. Furthermore different polymeric materialsshaped by different processes may be joined together to form a compositeshape. The composite shape can be a laminar structure. For example, apolymeric matrix may be attached to one or more polymeric matrixes toform a multilayer polymeric matrix structure. The attachment may beperformed by any suitable means such as gluing with a liquid polymer,stapling, suturing, or a combination of these methods. In addition, thepolymeric matrix may be formed as a solid block and shaped by laser orother standard machining techniques to its desired final form. Lasershaping refers to the process of removing materials using a laser.

The polymers can be characterized for mechanical properties such astensile strength using an Instron tester, for polymer molecular weightby gel permeation chromatography (GPC), glass transition temperature bydifferential scanning calorimetry (DSC) and bond structure by infrared(IR) spectroscopy; with respect to toxicology by initial screening testsinvolving Ames assays and in vitro teratogenicity assays, andimplantation studies in animals for immunogenicity, inflammation,release and degradation studies. In vitro cell attachment and viabilitycan be assessed using scanning electron microscopy, histology, andquantitative assessment with radioisotopes.

Substrates can be treated with additives or drugs prior to implantation(before or after the polymeric substrate is seeded with cells), e.g., topromote the formation of new tissue after implantation. Thus, forexample, growth factors, cytokines, extracellular matrix components, andother bioactive materials can be added to the substrate to promote grafthealing and formation of new tissue. Such additives will in general beselected according to the tissue or organ being reconstructed oraugmented, to ensure that appropriate new tissue is formed in theengrafted organ or tissue (for examples of such additives for use inpromoting bone healing, see, e.g., Kirker-Head, C. A. Vet. Surg. 24 (5):408-19 (1995)). For example, vascular endothelial growth factor (VEGF,see, e.g., U.S. Pat. No. 5,654,273 herein incorporated by reference) canbe employed to promote the formation of new vascular tissue. Growthfactors and other additives (e.g., epidermal growth factor (EGF),heparin-binding epidermal-like growth factor (HBGF), fibroblast growthfactor (FGF), cytokines, genes, proteins, and the like) can be added inamounts in excess of any amount of such growth factors (if any) whichmay be produced by the cells seeded on the substrate. Such additives arepreferably provided in an amount sufficient to promote the formation ofnew tissue of a type appropriate to the tissue or organ, which is to berepaired or augmented (e.g., by causing or accelerating infiltration ofhost cells into the graft). Other useful additives include antibacterialagents such as antibiotics.

The biocompatible substrate may be shaped using methods such as, solventcasting, compression molding, filament drawing, meshing, leaching,weaving and coating. In solvent casting, a solution of one or morepolymers in an appropriate solvent, such as methylene chloride, is castas a branching pattern relief structure. After solvent evaporation, athin film is obtained. In compression molding, the substrate is pressedat pressures up to 30,000 pounds per square inch into an appropriatepattern. Filament drawing involves drawing from the molten polymer andmeshing involves forming a mesh by compressing fibers into a felt-likematerial. In leaching, a solution containing two materials is spreadinto a shape close to the final form of the tissue. Next a solvent isused to dissolve away one of the components, resulting in poreformation. (See Mikos, U.S. Pat. No. 5,514,378, hereby incorporated byreference).

In nucleation, thin films in the shape of the tissue are exposed toradioactive fission products that create tracks of radiation damagedmaterial. Next, the polycarbonate sheets are etched with acid or base,turning the tracks of radiation-damaged material into pores. Finally, alaser may be used to shape and burn individual holes through manymaterials to form a tissue structure with uniform pore sizes. Thesubstrate can be fabricated to have a controlled pore structure thatallows nutrients from the culture medium to reach the deposited cellpopulation. In vitro cell attachment and cell viability can be assessedusing scanning electron microscopy, histology and quantitativeassessment with radioisotopes.

Thus, the substrate can be shaped into any number of desirableconfigurations to satisfy any number of overall system, geometry orspace restrictions. The matrix can be shaped to different sizes toconform to the necessary structures of different sized patients.

A substrate can also be permeated with a material, for example liquifiedcopolymers (poly-DL-lactide co-glycolide 50:50 80 mg/ml methylenechloride) to alter its mechanical properties. This can be performed bycoating one layer, or multiple layers until the desired mechanicalproperties are achieved.

The substrate can also be treated or seeded with various factors andproteins to control the degradation/absorption of the matrix in thesubject. For instance, if the cells seeded within the substrate areslow-growing, then it is beneficial to maintain the matrix integrity fora long enough period of time to allow the cells enough time toregenerate and grow. On the other hand, if the cells are able to quicklyreproduce and grow, then a short lived substrate could be desirable.Varying the concentration of aprotinin additives, aminocaproic acid,tranxemic acid, or similar fibrinolytic inhibitors or the degree ofchemical cross-linking in the matrix could be used to precisely controlthis variable. The substrate could also be seeded with varying growthfactors such as angiogenesis factor to promote a growth of blood vesselsupon implantation.

VI. Functionalized Matrices

A matrix (e.g., an electrospun matrix, a natural decellularized matrix,or synthetic matrix) can be functionalized by incorporation ofnanoparticles such as quantum dots (QD) coupled to therapeutic orbiological agents. The matrix can also be functionalized to incorporatea contrast enhancing agent (e.g., gadolinium).

In one aspect, the invention pertains to releasing therapeutic orbiological agent in a controlled manner at a target site. This isaccomplished using quantum dots to which the therapeutic/biologicalagent is coupled. Quantum dots are a semiconductor nanocrystal withsize-dependent optical and electronic properties. In particular, theband gap energy of a quantum dot varies with the diameter of thecrystal. The average diameter of the QDs may be between about 1 to about100 nm, between about 10-80 nm, and between about 25-40 nm. The coupledagent can be released by application of energy such as near infrared(NIR) irradiation from a laser source, which causes the bonds betweenthe agent and the QD to break and thus releases the agent. This allowsthe release of the agent to be controlled by triggering its release uponapplication of energy. Quantum dots have been used as photostablebiological fluorescent tags, semiconductors, and thermal therapy. Thehigh transmission, scattering-limited attenuation, and minimal heatingeffects of quantum dots makes these suitable for the coupling oftherapeutic/biological agents. In one embodiment, NIR CdSe quantum dots(Evident Technologies) can be used. These QDs have an optical absorptionrange of 700-1000 nm. NIR energy within this spectral region has beenshown to penetrate tissue at depths up to 23 cm with no observabledamage to the intervening tissue.

A matrix functionalized with a QD coupled to a therapeutic or biologicalagent can be used for controlled release of the therapeutic orbiological agent at a target in the subject. The therapeutic orbiological agent can be released by application of energy at a desiredwavelength such as near infrared irradiation. Due to localized heatingof the QD, ultrastructural changes cause the release of the coupledagent. The release kinetics can be varied according to the type of QDused and the wavelength of irradiation. The release kinetics can also bevaried by altering the intensity and time of irradiation. For example, aQD (e.g., CdSe QD from Evident Technologies) coupled to encapsulatedheparin can be incorporated into an electrospun matrix. Upon applicationof near infrared radiation at a wavelength of 700-1000 nm, the heparinis released in a controlled manner, as described in the examples below.

The studies in the examples section demonstrate the burst release ofheparin over time when quantum dot conjugated heparin nanoparticles wereirradiated by NIR irradiation. This system allows medical personnel totune therapeutic/biological agent release rates post-operatively.

The emission spectra of quantum dots have linewidths as narrow as 25-30nm depending on the size heterogeneity of the sample, and lineshapesthat are symmetric, gaussian or nearly gaussian with an absence of atailing region. The combination of tunability, narrow linewidths, andsymmetric emission spectra without a tailing region provides for highresolution of multiply-sized quantum dots within a system and allowssimultaneous examination of a variety of biological moieties tagged withQDs.

In addition, the range of excitation wavelengths of the quantum dots isbroad and can be higher in energy than the emission wavelengths of allavailable quantum dots. Consequently, this allows the simultaneousexcitation of all quantum dots in a system with a single light source.The ability to control the size of QDs enables one to construct QDs withfluorescent emissions at any wavelength in the UV-visible-IR region.Therefore, the colors (emissions) of QDs are tunable to any desiredspectral wavelength. Furthermore, the emission spectra of monodisperseQDs have linewidths as narrow as 25-30 nm. The linewidths are dependenton the size heterogeneity of QDs in each preparation. In one embodiment,the QDs emit light in the ultraviolet wavelengths. In anotherembodiment, the QDs emit light in the visible wavelengths. In otherembodiments, the QDs emit light in the near-infrared and the infraredwavelengths. Color of the light emitted by the QDs may be size-tunableand excitation energy tunable.

Many QDs are constructed of elements from groups II-VI, III-V and IV ofthe periodic table. They exhibit quantum confinement effects in theirphysical properties, and can be used in the composition of theinvention. Exemplary materials suitable for use as quantum dots include,but are not limited to, ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, GaN, GaP,GaAs, GaSb, InP, InAs, InSb, AlS, AlP, AlAs, AlSb, PbS, PbSe, Ge, and Siand ternary and quaternary mixtures thereof. The quantum dots mayfurther include an overcoating layer of a semiconductor having a greaterband gap.

Any suitable therapeutic or biological agent such as genetic material,growth factors, cytokines, enzymes can be coupled to the QD. Thetherapeutic or biological agent can be released by the application ofenergy that breaks the bond between the QD and the coupled agent. Theagent may also be released at a specific site as a function ofbiodegradation of the matrix in the surrounding environment over time.

Examples of a therapeutic or biological agent include, but are notlimited to proteins growth factors, antibodies, nucleic acids molecules,carbohydrates, and the like. Growth factors useful in the presentinvention include, but are not limited to, transforming growthfactor-alpha (TGF-α), transforming growth factor-beta (TGF-β),platelet-derived growth factors (PDGF), fibroblast growth factors (FGF),including FGF acidic isoforms 1 and 2, FGF basic form 2 and FGF 4, 8, 9and 10, nerve growth factors (NGF) including NGF 2.5 s, NGF 7.0 s andbeta NGF and neurotrophins, brain derived neurotrophic factor, cartilagederived factor, bone growth factors (BGF), basic fibroblast growthfactor, insulin-like growth factor (IGF), vascular endothelial growthfactor (VEGF), granulocyte colony stimulating factor (G-CSF), insulinlike growth factor (IGF) I and II, hepatocyte growth factor, glialneurotrophic growth factor (GDNF), stem cell factor (SCF), keratinocytegrowth factor (KGF), transforming growth factors (TGF), including TGFsalpha, beta, beta1, beta2, beta3, skeletal growth factor, bone matrixderived growth factors, and bone derived growth factors and mixturesthereof.

Cytokines useful in the present invention include, but are not limitedto, cardiotrophin, stromal cell derived factor, macrophage derivedchemokine (MDC), melanoma growth stimulatory activity (MGSA), macrophageinflammatory proteins 1 alpha (MIP-1 alpha), 2, 3 alpha, 3 beta, 4 and5, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,IL-12, IL-13, TNF-alpha, and TNF-beta. Immunoglobulins useful in thepresent invention include, but are not limited to, IgG, IgA, IgM, IgD,IgE, and mixtures thereof. Some preferred growth factors include VEGF(vascular endothelial growth factor), NGFs (nerve growth factors),PDGF-AA, PDGF-BB, PDGF-AB, FGFb, FGFa, and BGF.

Other molecules useful as therapeutic or biological agents include, butare not limited to, growth hormones, leptin, leukemia inhibitory factor(LIF), endostatin, thrombospondin, osteogenic protein-1, bonemorphogenetic proteins 2 and 7, osteonectin, somatomedin-like peptide,osteocalcin, interferon alpha, interferon alpha A, interferon beta,interferon gamma, interferon 1 alpha.

Embodiments involving amino acids, peptides, polypeptides, and proteinsmay include any type or combinations of such molecules of any size andcomplexity. Examples include, but are not limited to structuralproteins, enzymes, and peptide hormones. These compounds can serve avariety of functions. In some embodiments, the matrix may containpeptides containing a sequence that suppresses enzyme activity throughcompetition for the active site. In other applications antigenic agentsthat promote an immune response and invoke immunity can be incorporatedinto a construct. In substances such as nucleic acids, any nucleic acidcan be present. Examples include, but are not limited todeoxyribonucleic acid (DNA), and ribonucleic acid (RNA). Embodimentsinvolving DNA include, but are not limited to, cDNA sequences, naturalDNA sequences from any source, and sense or anti-sense oligonucleotides.For example, DNA can be naked (e.g., U.S. Pat. Nos. 5,580,859;5,910,488) or complexed or encapsulated (e.g., U.S. Pat. Nos. 5,908,777;5,787,567). DNA can be present in vectors of any kind, for example in aviral or plasmid vector. In some embodiments, nucleic acids used willserve to promote or to inhibit the expression of genes in cells insideand/or outside the electrospun matrix. The nucleic acids can be in anyform that is effective to enhance its uptake into cells.

The state of the electrospun matrix in relation to the incorporatedtherapeutic or biological agent can be controlled by the couplingchemistry, whether the therapeutic/biological agent is encapsulated, theselection of matrix compounds, the type of QDs used, solvent(s), andsolubility of the matrix compounds in those solvents. These parameterscan be manipulated to control the release of the therapeutic/biologicalagents. It is to be understood that therapeutic/biological agents may beentrapped or entangled within an electrospun matrix, bonded to a matrix,contained within cavities, enclosures, inclusions, or pockets, orstructures of electrospun matrix (e.g. fibers, fibrils, particles) orexternally bound to specific sites on the matrix.

The therapeutic or biological agent can also be entrapped, for exampleencapsulated in a polymer with the QD. The encapsulated QD-agent can bemixed with a solution comprising at least one natural compounds, and atleast one synthetic compound and electrospun into the matrix.

In particular, the therapeutic or biological agent and the nanoparticles(e.g., quantum dot) can be entrapped or encapsulated to produce“nanocapsules.” These nanocapsules containing the agent and thenanoparticle can be produce standard encapsulating techniques.Microencapsulation of agents generally involves three steps: (a)generating microcapsules enclosing the agents (e.g., by forming dropletsof cell-containing liquid alginate followed by exposure to a solution ofcalcium chloride to form a solid gel), (b) coating the resulting gelledspheres with additional outer coatings (e.g., outer coatings comprisingpolylysine and/or polyornithine) to form a semipermeable membrane; and(c) liquefying the original core gel (e.g., by chelation using asolution of sodium citrate). The three steps are typically separated bywashings in normal saline.

Alginates are linear polymers of mannuronic and guluronic acid residues.Monovalent cation alginate salts, e.g., Na-alginate, are generallysoluble. Divalent cations such as Ca²⁺, Ba²⁺ or Sr²⁺ tend to interactwith guluronate, providing crosslinking and forming stable alginategels. Alginate encapsulation techniques typically take advantage of thegelling of alginate in the presence of divalent cation solutions.Alginate encapsulation of agent-nanoparticles generally involvessuspending the agent-nanoparticles to be encapsulated in a solution of amonovalent cation alginate salt, generating droplets of this solution,and contacting the droplets with a solution of divalent cations. Thedivalent cations interact with the alginate at the phase transitionbetween the droplet and the divalent cation solution, resulting in theformation of a stable alginate gel matrix being formed. A variation ofthis technique is reported in U.S. Pat. No. 5,738,876, where the cell issuffused with a solution of multivalent ions (e.g., divalent cations)and then suspended in a solution of gelling polymer (e.g., alginate), toprovide a coating of the polymer.

Another method of microencapsulating agent-nanoparticles is thealginate-polyamino acid technique. Cells are suspended in sodiumalginate in saline, and droplets containing islets are produced.Droplets of cell-containing alginate flow into calcium chloride insaline. The negatively charged alginate droplets bind calcium and form acalcium alginate gel. The microcapsules are washed in saline andincubated with poly-L-lysine (PLL) or poly-L-ornithine (or combinationsthereof); the positively charged poly-l-lysine and/or poly-L-ornithinedisplaces calcium ions and binds (ionic) negatively charged alginate,producing an outer poly-electrolyte membrane. A final coating of sodiumalginate may be added by washing the microcapsules with a solution ofsodium alginate, which ionically bonds to the poly-L-lysine and/orpoly-L-ornithine layer. See U.S. Pat. No. 4,391,909 to Lim et al (allU.S. patents referenced herein are intended to be incorporated herein intheir entirety). This technique produces what has been termed a“single-wall” microcapsule. Preferred microcapsules are essentiallyround, small, and uniform in size. (Wolters et al., J. Appli Biomater.3:281 (1992)).

The alginate-polylysine microcapsules can also then be incubated insodium citrate to solubilize any calcium alginate that has not reactedwith poly-l-lysine, i.e., to solubilize the internal core of sodiumalginate containing the islet cells, thus producing a microcapsule witha liquefied cell-containing core portion. See Lim and Sun, Science210:908 (1980). Such microcapsules are referred to herein as having“chelated”, “hollow” or “liquid” cores. A “double-wall” microcapsule isproduced by following the same procedure as for single-wallmicrocapsules, but prior to any incubation with sodium citrate, themicrocapsules are again incubated with poly-l-lysine and sodiumalginate.

Many alternative techniques used for encapsulating agents are known inthe art and can be used with this invention. U.S. Pat. No. 5,084,350discloses microcapsules enclosed in a larger matrix, where themicrocapsules are liquefied once the microcapsules are within the largermatrix. Tsang et al., U.S. Pat. No. 4,663,286 discloses encapsulationusing an alginate polymer, where the gel layer is cross-linked with apolycationic polymer such as polylysine, and a second layer formed usinga second polycationic polymer (such as polyornithine); the second layercan then be coated by alginate. U.S. Pat. No. 5,762,959 to Soon-Shionget al. discloses a microcapsule having a solid (non-chelated) alginategel core of a defined ratio of calcium/barium alginates, with polymermaterial in the core. U.S. Pat. Nos. 5,801,033 and 5,573,934 to Hubbellet al. describe alginate/polylysine microspheres having a finalpolymeric coating (e.g., polyethylene glycol (PEG)); Sawhney et al.,Biomaterials 13:863 (1991) describe alginate/polylysine microcapsulesincorporating a graft copolymer of poly-l-lysine and polyethylene oxideon the microcapsule surface, to improve biocompatibility; U.S. Pat. No.5,380,536 describes microcapsules with an outermost layer of watersoluble non-ionic polymers such as polyethylene(oxide). U.S. Pat. No.5,227,298 to Weber et al. describes a method for providing a secondalginate gel coating to cells already coated with polylysine alginate;both alginate coatings are stabilized with polylysine. U.S. Pat. No.5,578,314 to Weber et al. provides a method for microencapsulation usingmultiple coatings of purified alginate. U.S. Pat. No. 5,693,514 toDorian et al. reports the use of a non-fibrogenic alginate, where theouter surface of the alginate coating is reacted with alkaline earthmetal cations comprising calcium ions and/or magnesium ions, to form analkaline earth metal alginate coating. The outer surface of the alginatecoating is not reacted with polylysine. U.S. Pat. No. 5,846,530 toSoon-Shiong describes microcapsules containing cells that have beenindividually coated with polymerizable alginate, or polymerizablepolycations such as polylysine, prior to encapsulation.

In one embodiment, heparin is coupled to the nanoparticle and thecontrol the release kinetics of heparin can be monitored. One skilled inthe art will appreciate that the control release kinetics depend on thecapsulation parameters including nanocapsule size, heparin and quantumdot loading, and polymer composition. The mean diameter of thenanocapsules depends on the mixing velocity of the preparation processand viscosity of the preparation media. Nanocapsule size can be reducedby exposing the preparation to sonication over a range of about 30second to about 120 seconds, increasing the sonication intensity fromabout 5 watts to about 20 watts, or by varying the ratios of organicpolymer phase to aqueous heparin phase. Nanocapsule sizes can becharacterized by scanning electron microscopy (SEM), coulter counter,and light scattering.

In one embodiment, the heparin can be conjugated to quantum dots byusing an EDC/NHS chemical method. Various concentrations of heparin(ranging form 10-30 weight % polymer) and quantum dots can be used todetermine optimal loading efficiency.

For polymer encapsulation, FDA approved biodegradable polymers (PLA,PLGA, PCL) can be used for the control of encapsulation and degradationof the nanocapsules in vivo.

The examples show that a burst of heparin release occurs using abroadband infrared (IR) source. Using measured quantities of QD-Heparinnanocapsules (NC) suspended in a physiological buffer, the influence ofvarying wavelengths, intensities, and irradiation times on the releasekinetics can be determined. In one embodiment, the wavelength ofirradiation used on the QD-Heparin can be in the near-infraredwavelength range, such as 700 nm, 800 nm, and 900 nm, using a filteredxenon source. The intensity of irradiation energy can be adjusted inincremental steps from 0 (control), 1 mW/cm², 10 mW/cm², 100 mW/cm², 1W/cm², and 10 W/cm². The irradiation time can also be varied todetermine the optimal irradiation time at each effective powerintensity. The irradiation time can vary from 0 (control), 10, 60, 300,and 600 seconds of exposure.

The encapsulated QD-heparin will be released upon near infra-red (NIR)irradiation due to localized heating of the quantum dots which inducesultrastructural changes in the nanocapsules. The release kinetics willbe varied at the target site by modulating the intensity and time of NIRirradiation to produce a controlled release of heparin. The quantitativemeasurement of heparin released from the nanocapsules can be measuredover time (2, 4, 6, 12, and 24 hours and daily thereafter up to 30 days)and measured for its anti-factor Xa activity with a syntheticchromogenic substrate using a kit Rotachrom (Diagnostica Stago Inc).

In another aspect, the invention pertains to monitoring tissueremodeling a tissue engineered construct. Remodeling that takes placetoo slowly can result in pathologic response of surrounding tissues andcompliance mismatch of the vessel. Rapid remodeling can result inpremature failure of the engineered construct. Magnetic ResonanceImaging (MRI) is a powerful, non-invasive technique that can be usedlong term for monitoring the remodeling process. Nanoparticles (e.g.,QD, image enhancing agents) can be easily bound both to decellularizedmatrices and electrospun matrices, and also embedded within nanofibersof electrospun matrices. The nanoparticles provide high MRI contrast,and due to their small size, will not interfere with normal biologicalprocesses. Organolanthanide complexes containing paramagnetic metalssuch as gadolinium (Gd) have been known to cause distortion in anelectromagnetic field. When the protons in water interact with thisdistorted field, their magnetic properties significantly change suchthat they can be detected by MRI. The Examples demonstrate the enhancedimaging observed using MRI contrast with Gd functionalized nanoparticlesbound to the surface and/or incorporated into the vascular matrices ornanocapsules. Other examples of contrast enhancing agents include, butare not limited to, rare earth metals such as, cerium, samarium,terbium, erbium, lutetium, scandium, barium, bismuth, cerium,dysprosium, europium, hafnium, indium, lanthanum, neodymium, niobium,praseodymium, strontium, tantalum, ytterbium, yttrium, and zirconium.

In one embodiment, the agents are joined to the matrix by peptide bonds.For example, nanoparticles can be incorporated as part of the matrixusing EDC (1-ethyl-3(3-dimethly aminopropyl)carbodiimide) andsulfo-NHS(N-hydrocyl-sulfo-succinimide) to form peptide bonds. Variousother know techniques can be used as described, for example, inHeumanson, Bioconjugate Techniques, Academic Press San Diego, Calif.,1996, herein incorporated by reference. For external functionalization,a peptide bond can be created between the matrix and carboxylatedgadolinium nanoparticles using the EDC/sulpho-NHS method to form peptidebonds between the carboxylates and amino groups. The quantum dot coupledto a therapeutic/biological agent, a contrast enhancing agent, e.g.,gadolinium, or both, can also be added internally to an electrospunmatrix by incorporating each component into the solution with at leastone natural compound and at least one synthetic compound. For example,solutions containing collagen I, elastin and PLGA, successfullyincorporated the contrast enhancing agent gadolinium uponelectrospinning as described in the Examples. The incorporation of thegadolinium into the matrix can be observed in vitro and in vivo usingdetection methods such as magnetic resonance imaging (MRI). Thus, amatrix functionalized with a contrasting agent allows the degradation ofthe matrix to be monitored.

Any type of functionalization method can be used. Examples of somepossible functionalization chemistries include, but are not limited to,esterification (e.g., with acyl halides, acid anhydrides, carboxylicacids, or esters via interchange reactions), ether formation (forexample, via the Williamson ether synthesis), urethane formation viareactions with isocyanates, sulfonation with, for example,chlorosulfonic acid, and reaction of b-sulfato-ethylsulfonyl aniline toafford an amine derivative that can be converted to a diazo for reactionwith a wide variety of compounds. Such chemistries can be used to attacha wide variety of substances to the electrospun matrix, including butnot limited to crown ethers (Kimura et al., (1983) J. Polym. Sci. 21,2777), enzymes (Chase et al. (1998) Biotechnol. Appl. Biochem., 27,205), and nucleotides (Overberger et al. (1989) J. Polym. Sci. 27,3589).

V. Culturing Cells

The artificial tissue can be created by using allogenic cell populationsderived from the subject's own tissue. The artificial tissue can also bexenogenic, where cell populations are derived from a mammalian speciesthat are different from the subject. For example, tissue cells can bederived from mammals such as monkeys, dogs, cats, mice, rats, cows,horses, pigs, goats and sheep.

The isolated cells are preferably cells obtained by a swab or biopsy,from the subject's own tissue. A biopsy can be obtained by using abiopsy needle under a local anesthetic, which makes the procedure quickand simple. The small biopsy core of the isolated tissue can then beexpanded and cultured to obtain the tissue cells. Cells from relativesor other donors of the same species can also be used with appropriateimmunosuppression.

Methods for the isolation and culture of cells are discussed byFreshney, Culture of Animal Cells. A Manual of Basic Technique, 2d Ed.,A. R. Liss, Inc., New York, 1987, Ch. 9, pp. 107-126. Cells may beisolated using techniques known to those skilled in the art. Forexample, the tissue can be cut into pieces, disaggregated mechanicallyand/or treated with digestive enzymes and/or chelating agents thatweaken the connections between neighboring cells making it possible todisperse the tissue into a suspension of individual cells withoutappreciable cell breakage. If necessary, enzymatic dissociation can beaccomplished by mincing the tissue and treating the minced tissue withany of a number of digestive enzymes either alone or in combination.These include but are not limited to trypsin, chymotrypsin, collagenase,elastase, and/or hyaluronidase, DNase, pronase, and dispase. Mechanicaldisruption can also be accomplished by a number of methods including,but not limited to, scraping the surface of the tissue, the use ofgrinders, blenders, sieves, homogenizers, pressure cells, or insonatorsto name but a few.

Cell types include, but are not limited to, endothelial cells such ashuman endothelial cells, progenitor cells isolated from the peripheralblood bone that can be induced to differentiate into different cells,stem cells, committed stem cells, and/or differentiated cells may beused. Also, depending on the type of tissue or organ being made,specific types of committed stem cells can be used. For instance,myoblast cells can be used to build various muscle structures. Othertypes of committed stem cells can be used to make organs or organ-liketissue such as heart, kidney, liver, pancreas, spleen, bladder, ureterand urethra. Other cells include, but are not limited to, endothelialcells, muscle cells, smooth muscle cells, fibroblasts, osteoblasts,myoblasts, neuroblasts, fibroblasts, glioblasts; germ cells,hepatocytes, chondrocytes, keratinocytes, cardiac muscle cells,connective tissue cells, epithelial cells, endothelial cells,hormone-secreting cells, cells of the immune system, neurons, cells fromthe heart, kidney, liver, pancreas, spleen, bladder, ureter and urethra,and the like. In some embodiments it is unnecessary to pre-select thetype of stem cell that is to be used, because many types of stem cellscan be induced to differentiate in an organ specific pattern oncedelivered to a given organ. For example, a stem cell delivered to theliver can be induced to become a liver cell simply by placing the stemcell within the biochemical environment of the liver.

Examples also include cells that have been genetically engineered,transformed cells, and immortalized cells. One example of geneticallyengineered cells useful in the present invention is a geneticallyengineered cell that makes and secretes one or more desired molecules.When matrices comprising genetically engineered cells are implanted inan organism, the molecules produced can produce a local effect or asystemic effect, and can include the molecules identified above aspossible substances.

Cells may produce substances that inhibit or stimulate inflammation;facilitate healing; resist immunorejection; provide hormone replacement;replace neurotransmitters; inhibit or destroy cancer cells; promote cellgrowth; inhibit or stimulate formation of blood vessels; augment tissue;and to supplement or replace the following tissue, neurons, skin,synovial fluid, tendons, cartilage, ligaments, bone, muscle, organs,dura, blood vessels, bone marrow, and extracellular matrix.

The shape of the extracellular matrix may help send signals to the cellsto grow and reproduce in a specific type of desired way. Other factorsand differentiation inducers may be added to the matrix to promotespecific types of cell growth.

Once the tissue has been reduced to a suspension of individual cells,the suspension can be fractionated into subpopulations from which thecells elements can be obtained. This also may be accomplished usingstandard techniques for cell separation including, but not limited to,cloning and selection of specific cell types, selective destruction ofunwanted cells (negative selection), separation based upon differentialcell agglutinability in the mixed population, freeze-thaw procedures,differential adherence properties of the cells in the mixed population,filtration, conventional and zonal centrifugation, centrifugalelutriation (counterstreaming centrifugation), unit gravity separation,countercurrent distribution, electrophoresis and fluorescence-activatedcell sorting (see e.g., Freshney, (1987) Culture of Animal Cells. AManual of Basic Techniques, 2d Ed., A. R. Liss, Inc., New York, Ch. 11and 12, pp. 137-168). For example, salivary cells may be enriched byfluorescence-activated cell sorting. Magnetic sorting may also be used.

Cell fractionation may also be desirable, for example, when the donorhas diseases such as cancer or tumor. A cell population may be sorted toseparate the cancer or tumor cells from normal noncancerous cells. Thenormal noncancerous cells, isolated from one or more sorting techniques,may then be used for tissue reconstruction.

Isolated cells can be cultured in vitro to increase the number of cellsavailable for seeding into the biocompatible substrate. To prevent animmunological response after implantation of the artificial tissueconstruct, the subject may be treated with immunosuppressive agents suchas, cyclosporin or FK506.

Isolated cells may be transfected with a nucleic acid sequence. Usefulnucleic acid sequences may be, for example, genetic sequences whichreduce or eliminate an immune response in the host. For example, theexpression of cell surface antigens such as class I and class IIhistocompatibility antigens may be suppressed. In addition, transfectioncould also be used for gene delivery. Cells may be transfected withspecific genes prior to seeding onto the biocompatible substitute. Thus,the cultured cells can be engineered to express gene products that wouldproduce a desired protein that helps ameliorate a particular disorder.

The tissue cells grown on the electrospun matrix substrate may begenetically engineered to produce gene products beneficial toimplantation, e.g., anti-inflammatory factors, e.g., anti-GM-CSF,anti-TNF, anti-IL-1, and anti-IL-2. Alternatively, the tissue cells maybe genetically engineered to “knock out” expression of native geneproducts that promote inflammation, e.g., GM-CSF, TNF, IL-1, IL-2, or“knock out” expression of MHC in order to lower the risk of rejection.

Methods for genetically engineering cells for example with retroviralvectors, adenoviral vectors, adeno-associated viral vectors,polyethylene glycol, or other methods known to those skilled in the artcan be used. These include using expression vectors which transport andexpress nucleic acid molecules in the cells. (See Geoddel; GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990).

Vector DNA is introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. Suitable methodsfor transforming or transfecting host cells can be found in Sambrook etal. Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold SpringHarbor Laboratory press (1989), and other laboratory textbooks.

Once seeded onto the matrix, the cells will proliferate and develop onthe matrix to form a tissue layer. Importantly, because the matrix hasan infra-structure that permits culture medium to reach the tissuelayer, the cell population continues to grow, divide, and remainfunctionally active to develop into a tissue that has a morphology whichresembles the analogous structure in vivo.

It is important to recreate, in culture, the cellular microenvironmentfound in vivo for the particular tissue being engineered. By using amatrix that retains an infra-structure that is similar or the same as anin vivo tissue structure, the optimum environment for cell-cellinteractions, development and differentiation of cell populations, iscreated.

Growth factors and regulatory factors can be added to the media toenhance, alter or modulate proliferation and cell maturation anddifferentiation in the cultures. The growth and activity of cells inculture can be affected by a variety of growth factors such as growthhormone, somatomedins, colony stimulating factors, erythropoietin,epidermal growth factor, hepatic erythropoietic factor (hepatopoietin),and like. Other factors which regulate proliferation and/ordifferentiation include prostaglandins, interleukins, andnaturally-occurring chalones.

The artificial tissue constructs of the invention can be used in avariety of applications. For example, the artificial tissue constructscan be implanted into a subject to replace or augment existing tissue.The subject can be monitored after implantation of the artificial tissueor organ, for amelioration of the disorder.

The artificial tissue can be used in vitro to screen a wide variety ofcompounds, for effectiveness and cytotoxicity of pharmaceutical agents,chemical agents, growth/regulatory factors. The cultures can bemaintained in vitro and exposed to the compound to be tested. Theactivity of a cytotoxic compound can be measured by its ability todamage or kill cells in culture. This may readily be assessed by vitalstaining techniques. The effect of growth/regulatory factors may beassessed by analyzing the cellular content of the matrix, e.g., by totalcell counts, and differential cell counts. This may be accomplishedusing standard cytological and/or histological techniques including theuse of immunocytochemical techniques employing antibodies that definetype-specific cellular antigens. The effect of various drugs on normalcells cultured in the artificial tissue may be assessed.

VI. Preconditioning Chamber

Blood vessels can be created in a preconditioning chamber as shown inFIG. 11, which is one embodiment of a preconditioning chamber. Thedimensions of the chamber are such that it can hold a matrix seeded withcells. Suitable size ranges can range form about 200×200×600 mm,100×100×300 mm, preferably about 50×50×150 mm. FIG. 11A shows the topview of one embodiment of the preconditioning chamber 40 with a firstand second long wall 42 and a first and second short wall 44. The firstand second short walls 44 each have an opening 46 that accommodates aplatform 48 that can hold a vessel 50 having an attachment element 52.The matrix seeded with cells (shown in hashed lines) can be attached tothe first and second attachment ends 53 of the vessel. The vessel isoperatively linked to a fluid flow system (not shown) that can pumpbiological fluid through one end of the vessel 50, through the attachedtubular matrix seeded with cells, and through the other end of thevessel in a continuous manner. FIG. 11B shows the side view of thepreconditioning chamber of FIG. 11A and FIG. 11C shows a traverse viewFIG. 11A.

The biological fluid can be pumped using any pumping mechanism such as agear pump. The chamber can further comprise a rotation device that canbe used to rotate the chamber at a desired angle for example, by 45°,90°, 180°, and 360°. The rotation device can be manually operated or canbe automated such that the chamber is rotated at a desired speed and ata desired time. In other embodiments, the chamber can be amultichambered and can house more than one blood vessel. In otherembodiments, both the inside and the outside of the seeded matrix can bepreconditioned using the preconditioning chamber of the invention. Insuch embodiments, the chamber is filled with a volume of preconditioningfluid that can cover the attached seeded matrix. The fluid flow of thebiological fluid on the outside of the matrix can be controlled by thesame or a separate mechanism than the fluid flow on the inside of thematrix. The biological fluid on the outside may be the same as thebiological fluid on the inside. Alternatively, or the biological fluidon the outside may be the different than the biological fluid on theinside. The fluid flow parameters an be the same for the biologicalfluid on the inside and the outside, or can be different.

The walls of the preconditioning chamber can be made of any suitablematerial such as plexiglass, plastics and the like as long as thematerial does not react with a biological fluid. The biological fluidcan be moved through the inside surface (lumen) of the attached matrixas a continuous flow, for example with a continuous flow-rate that canbe incremented over time to induce a wall shear in the range of about 1dyne/cm² to about 30 dynes/cm². The step of preconditioning the matrixcan also involve moving the biological fluid through the inside surfaceof the attached matrix as a pulsed flow, for example, a pulsed flow thathas a pulse-rate which is incremented over time to induce a wall shearin the range of about 10 dynes/cm² to about 45 dynes/cm². The pulse-ratecan be incremented over time to induce a wall pressure distribution inthe engineered blood vessel in the range of about 60 to about 200 mmHg.A different of the same biological fluid can also be used toprecondition the outside of the matrix.

The biological fluid can have a composition and viscosity that mimicsblood so that the engineered blood vessels are exposed same fluid flowdynamics as native blood vessels. Examples of biological fluids caninclude any buffer, medium of physiological fluid (e.g., DMEM with 10%FCS) The viscosity of the fluids can be altered by adding high molecularweight proteins such as 100 kDa dextran. Other molecular weight dextranscan also be used. It will be appreciated that the amount of dextran tobe used depends on the molecular weight and can range from about 10%,20%, 30%, 40%, 50%, and 60%. The composition may also be varied byadding other blood like constituents such as salts.

VII. Use of Matrices

The methods and compositions of the invention can be used for localizeddelivery of therapeutic/biological agents, as well as controlled releaseof such agents at the target site in a subject.

(i) Vascular Constructs

The methods and compositions of the invention can be used to constructblood vessels. One application of the electrospun matrices ordecellularized matrices is in the formation of medium and small diametervascular constructs. Some preferred materials for this embodiment arecollagen and elastin, especially collagen type I and collagen type III.Examples of vascular constructs include, but are not limited to coronaryvessels for bypass or graft, femoral artery, popliteal artery, brachialartery, tibial artery, radial artery or corresponding veins. Theelectrospun material is useful especially when combined with endothelialcells and smooth muscle cells. More complicated shapes including taperedand/or branched vessels can also be constructed. A different-shapedmandrel is necessary to wind the large fibers around or to orient theelectrospun polymer.

Some of the complications with vascular matrices are (1) thrombusformation and (2) inability to quantitatively monitor integration of thevascular graft in vivo. Problems with thrombus formation are some of themost difficult challenges resulting in frequent failure of vasculargrafts. Heparin, a powerful anticoagulation agent, is commonlyadministered clinically to avoid thrombus formation. However, systemicuse of heparin carries a certain amount of risk, thus locallyadministered heparin is preferred. The methods and compositions of theinvention can be used to overcome the lack of control of drug release byutilizing quantum dot based nanotechnology. Specifically, theacceleration of release of anticoagulants such as heparin at the targetlocation (vascular graft) by triggering their release from quantum dotsusing NIR energy. This allows the release kinetics of the anticoagulante.g., heparin to be modulated.

The studies shown in the Examples section demonstrated that nearinfrared (NIR) quantum dot conjugated heparin can be successfullyincorporated into the nanoparticles and vascular scaffolds to enable thecontrolled release (or burst release) of heparin over time initiated bynear infrared exposure.

MRI contrasting agents such as gadolinium were also successfullyattached to, or incorporated into the scaffold to enhance visualization.Thus, controlled release of heparin from vascular scaffolds can beachieved using near infrared (NIR) quantum dots and heparin and (2)nanocontrast agents functionalized on, or incorporated into, thevascular scaffold can be used to evaluate and monitor heparin release.

(ii) Tissue Organ Constructs

The methods and compositions of the invention can be used to constructengineered tissue organ constructs, or parts of organ constructs e.g.,heart, heart valves, liver, kidney, and the like. The ability to useelectrospun materials and matrices to bioengineer tissue or organscreates a wide variety of bioengineered tissue replacement applications.Examples of bioengineered components include, but are not limited to,blood vessels, heart, liver, kidney, skeletal muscle, cardiac muscle,and nerve guides. In some embodiments, such matrices are combined withtherapeutic agents that improve the function of the implant. Forexample, antibiotics, anti-inflammatories, local anesthetics orcombinations thereof, can be added to the matrix of a bioengineeredorgan to speed the healing process and reduce discomfort.

(iii) Substance Delivery

The methods and compositions of the invention can be used to deliveryone or more therapeutic agents to a desired location. The presentcompositions can be used to deliver therapeutic agents to an in vivolocation, an in vitro location, or other locations. The presentcompositions can be administered to these locations using any method.Alternatively, an electrospun matrix containing cells can be implantedin a body and used to deliver molecules produced by the cells afterimplantation.

The selection of the therapeutic agent and the method by which the agentis combined with the electrospun material affects the substance releaseprofile. To the extent that the agents are immobilized by theelectrospun matrix, the release rate is more closely related to the rateat which the electrospun material degrades. For example, a therapeuticagent can be electrospun with the matrix and trapped within anelectrospun filaments, in such an instance, the release kinetics aredetermined by the rate at which the electrospun matrix degrades. Inother embodiment, the therapeutic agent can be coupled to a QD andelectrospun into the matrix. In such instances, the release kinetics arecontrolled by the application of irradiation energy that disrupts thecoupling bonds between the therapeutic agent and the QD to release thetherapeutic agent. In other embodiments, the therapeutic agents can beencapsulated within a polymer matrix and the encapsulated therapeuticagent added during the electrospinning process such that theencapsulated therapeutic agents is embedded within the matrix. Underthese circumstances, the release kinetics depend on the rate at whichthe electrospun matrix degrades, as well as the nature and degradationproperties of the encapsulating polymer. In yet other embodiments, thetherapeutic agent can be coupled to a quantum dot and encapsulated andthen electrospun to become embedded within the matrix. Under suchcircumstances, the release kinetics are controlled by the application ofirradiation energy that disrupts the coupling bonds between thetherapeutic agent and the QD to release the therapeutic agent. Theporosity of the electrospun material can also be regulated, whichaffects the rate of release of a substance.

Chemicals that affect cell function, such as oligonucleotides, promotersor inhibitors of cell adhesion, hormones, and growth factors, forexample, can be incorporated into the electrospun matrix and the releaseof those substances from the electrospun matrix can provide a means ofcontrolling expression or other functions of cells in the electrospunmatrix.

Release kinetics in some embodiments are manipulated by cross-linkingelectrospun material through any means. In some embodiments,cross-linking will alter, for example, the rate at which the electrospunmatrix degrades or the rate at which a compound is released from theelectrospun matrix by increasing structural rigidity and delayingsubsequent dissolution of the electrospun matrix. Electrospun matrix canbe formed in the presence of cross-linking agents or can be treated withcross-linking agents after electrospinning. Any technique forcross-linking materials may be used as known to one of ordinary skill inthe art. Examples of cross-linking agents include, but are not limitedto, condensing agents such as aldehydes e.g., glutaraldehyde,carbodiimide EDC (1-ethyl-3(3 dimethyl aminopropyl)), photosensitivematerials that cross-link upon exposure to specific wavelengths oflight, osmium tetroxide, carbodiimide hydrochloride, and NHS(n-hydroxysuccinimide).

The release kinetics of the matrix is also controlled by manipulatingthe physical and chemical composition of the electrospun matrix. Forexample, small fibers of PLGA are more susceptible to hydrolysis thanlarger diameter fibers of PLGA. An agent delivered within an electrospunmaterial composed of smaller PLGA fibers is released more quickly thanwhen prepared within a matrix composed of larger diameter PLGA fibers.

Physical processing of the electrospun matrix is another way tomanipulate release kinetics. In some embodiments, mechanical forces,such as compression, applied to an electrospun matrix hasten thebreakdown of the matrix by altering the crystalline structure of thematrix. The structure of the matrix is thus another parameter that canbe manipulated to affect release kinetics. Polyurethanes and otherelastic materials such as poly(ethylene-co-vinyl acetate), silicones,and polydienes (e.g., polyisoprene), polycaprolactone, polyglycolic acidand related polymers are examples of materials whose release rate can bealtered by mechanical strain.

VIII. Storage

A matrix can be stored and used shortly before implantation by seedingit with cells. Many electrospun matrices are dry once they are spun andcan be storage in a dry or frozen state. Storage conditions will dependon the electrospun compounds used and whether a therapeutic agent isincorporated onto or into the matrix. In embodiments where a therapeuticagent is incorporated, the matrix can be stored at temperatures below 0°C., under vacuum, or in a lyophilized state. Other storage conditionscan be used, for example, at room temperature, in darkness, in vacuum orunder reduced pressure, under inert atmospheres, at refrigeratortemperature, in aqueous or other liquid solutions, or in powdered formdepending on the materials in and on the matrix.

The matrices may be sterilized through conventional means known to oneof ordinary skill in the art such as radiation, and heat. The matricescan also be combined with bacteriostatic agents, such as thimerosal, toinhibit bacterial growth. In some embodiments, the compositions can betreated with chemicals, solutions, or processes that confer stability instorage and transport.

Other embodiments and used of the invention will be apparent to thoseskilled in the art from consideration of the specification and practiceof the invention disclosed herein. All U.S. patents and other referencesnoted herein for whatever reason are specifically incorporated byreference. The specification and examples should be considered exemplaryonly with the true scope and spirit of the invention indicated by theclaims.

EXAMPLES Example 1 Methods and Materials

Materials

Unless otherwise stated, all chemicals were purchased from Sigma (St.Louis, Mo.). ECL buffers and 125I-Sodium were purchased from PerkinElmerNEN (Boston, Mass.). Collagenase type II (1 mg/ml) was purchased fromBoehringer Mannheim (Mannheim, Germany). Endothelial medium (EBM-2) waspurchased from Cambrex Bio Science (Walkersville, Md.). PCR reagents andprimers, M199 medium, fetal bovine serum (FBS) and penicillin werepurchased from Life Technologies (Gaithersburg, Md.). Basic FibroblastGrowth Factor (bFGF) was a gift from Judith Abraham, (Scios Nova,Calif.). FITC-labeled monoclonal anti human CD31 antibodies werepurchased from Santa Cruz (Santa Cruz, Calif.). Rabbit polyclonal antivonWillebrand factor (vWF), anti smooth muscle actin antibodies, andFITC labeled anti-rabbit IgG antibodies were purchased from DAKO(Glostrup, Denmark). Monoclonal anti human CD31, Goat polyclonal anti-VECadherin (VE-Cad), Goat anti-vWF and Rabbit polyclonal anti Flk-1antibodies were purchased from Santa Cruz (Santa Cruz, Calif.). FITC andBiotin-labeled Ulex europeaus I lectin was purchased from Sigma (St.Louis, Mo.). Biotin-labeled goat anti-mouse IgG and biotin-labeled mouseanti-goat IgG were purchased from Santa Cruz (Santa Cruz, Calif.).Anti-CD105 antibodies were purchased from BD Pharmingen (San Diego,Calif.). FITC-conjugated avidin was purchased from Vector Laboratories(Burlingame, Calif.). Athymic mice were purchased from Jackson Labs (BarHarbor, Me.).

Methods

(i) Scaffold Preparation

Electrospun nanofiber scaffolds have been developed using a solution ofcollagen type I, elastin, and poly(D,L-lactide-co-glycolide) (PLGA, mol.ratio 50:50, Mw 110,000) (Boeringer-Ingelheim, Germany). Collagen type Ifrom calf skin (Elastin Products Company, Owensville, Mo.), elastin fromligamentum nuchae (bovine neck ligament), (Elastin Products Company,Owensville, Mo.), and PLGA are mixed at a relative concentration byweight of 45% collagen, 40% PLGA, and 15% elastin. The solutes aredissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (99+%) (Sigma ChemicalCompany, St. Louis, Mo.) at a total solution concentration of 15% (w/v)(150 mg/mL). High molecular weight PLGA, previously used forelectrospinning tissue scaffolds is added to the solution to increasemechanical strength of the scaffold and increase viscosity and spinningcharacteristics of the solution.

Physically, the electrospinning method requires a high voltage powersupply, a syringe pump, a polymer solution or melt to be spun, and agrounded collection surface. During electrospinning, the groundedmandrel rotates while the stage translates to ensure even deposition offibers onto the mandrel surface. Solutions were electrospun using a highvoltage power supply (Spellman High Voltage, Hauppauge, N.Y.) at 25 kVpotential between the solution tip and the grounded surface. Thesolution was delivered with a 5 mL syringe through an 18 gauge blunt tipneedle at a flow rate of 3.0 mL/hr using a syringe pump. Fibers collectonto a grounded mandrel at a distance of 15 cm from the tip. The mandrelis a 303 stainless steel rod which is rotated at ˜500 rpm. The mandrelsize is initially 4.75 mm to allow for contraction of the graft due tocrosslinking. Uniform scaffolds of 120 mm length were created using 2.4mL of solution. This apparatus is shown schematically in FIG. 1.

Scaffolds were further crosslinked for increased stability and strength,using two crosslinking methods. The scaffolds were soaked for twominutes in 20% dextran solution in phosphate buffered saline prior tocrosslinking to reduce hydration-induced swelling and contraction of thescaffold. The scaffolds were crosslinked by immersion in 1) 1%glutaraldehyde solution and 2) EDC/NHS in MES/EtOH solution for 2 hoursat room temperature. These data show that it is possible to fabricatevascular scaffolds from biological polymers with mechanics and structuresimilar to decellularized scaffolds and native arteries.

FIG. 1 shows the electrospinning apparatus in which fibers deposit ontoa grounded collection surface as solvent evaporates due to increasingsurface area/volume ratio of solution. The electrostatic field causessplaying of solution, and solutions of sufficient viscosity and surfacetension form fibrous mats which adhere to grounded surfaces.

(ii) Cell Seeding

A confluent monolayer of endothelial cells is the most important barrieragainst thrombus formation, and endothelial cell mediated NO productionis important to maintain vascular tone. Cells were seeded with a mouseendothelial cell line MS1 cells. The cells routinely cultured in tissueculture polystyrene flasks at 37° C. under 5% CO2 atmosphere wereharvested after the treatment with 0.1% trypsin-EDTA. The scaffolds weremounted in tissue culture dishes. After equilibration with PBS, thecells (1×10⁵/mL) were seeded to the scaffolds. The culture medium usedwas DMEM medium containing 10% FBS, and antibiotics. After 2 daysculture, the cell attachment was assessed using scanning electronmicroscopy.

(iii) Microscopy

The relative quantity and distribution of collagen and elastin in avascular scaffold is important to the mechanical properties and functionof the seeded graft (FIG. 1). To determine the distribution ofcomponents of the scaffolds, histo- and immunohistochemical analyseswere performed to identify collagen and elastin distribution.

(iv) Biocompatibility Testing (Cell Viability and Proliferation)

Long-term viability of cells is necessary for the seeded scaffold toremodel itself into a viable, patent vessel. Standard methods wereemployed to assess viability and proliferation. To test for cellviability, constructs were placed in 24-well plates with approximately100 mg of material per well. Four different types of material weretested for biocompatibility and cell survival, with one negative controlwell with no material: (1) GA-NFS (1% glutaraldehyde crosslinkedelectrospun scaffold); (2) EDC-NFS (EDC-crosslinked electrospunscaffold); (3) nBV (natural blood vessel, decellularized); (4) Latex(latex rubber, positive control).

Endothelial cells were seeded in the wells on a scaffold for testing viathe direct contact method. For cell viability, cell layers were rinsedwith PBS. 0.005% (w/v) neutral red was added in culture medium. Theneutral red solution was removed after 4 hours incubation at 37° C. with1% acetic acid and 50% ethanol solution by volume was added for dyeextraction, and dye extraction was shaken for 5 minutes. Absorbance wasthen measured at 540 nm using a spectrophotometer. The intensity of redcolor obtained was directly proportional to the viability of the cellsand inversely proportional to the toxicity of the material.

Cell proliferation was tested using the mitochondrial metabolic activityassay. Cell layers were first rinsed with PBS. MTT solution was added at1 mg/mL in PBS containing 1 mg/mL glucose. MTT solution was removedafter 4 hours incubation at 37° C. Dimethyl sulfoxide (DMSO) was used todissolve insoluble formazan crystals, and the absorbance at 540 nm wasmeasured using a spectrophotometer. The intensity of blue color wasdirectly proportional to the metabolic activity of the cell populationsand inversely proportional to the toxicity of the material or extract.

(v) Mechanical Testing

Compliance mismatch is one of the most common causes of vascular graftfailure, resulting in intimal hyperplasia and occlusion. If the scaffoldis too compliant, it may form an aneurysm.

Scaffolds were immersed in a water bath and cannulated at either end.One cannula was connected to a column of water and the other to adrainage tube. The column of water was high enough to create a pressurewithin the vessel-shaped scaffold of 120 mmHg. Water was drained throughthe scaffold in order to lower the pressure in increments of 10 mmHg. Ateach increment, the diameter of the scaffold was recorded using adigital camera. This process was repeated until the pressure was 0 mmHg.

(vi) Axial and Circumferential Segment Testing

Vessels must resist higher stress in the circumferential direction thanin the axial direction. Native vessels adapt their mechanics to thisloading environment. It is important that the electrospun scaffoldsexhibit a mechanical strength at least that of native vessels.Mechanical loading tests were performed on the electrospun vessels inthe axial and circumferential directions using a uniaxial load testmachine (Instron Corporation, Issaquah, Wash.). A short segment from atubular scaffold was clamped at its cut ends for the axial test. Thecrosshead speed was set at 0.5 mm/sec and the test was stopped when thestrain decreased by 10% after the onset of failure. For testing in thecircumferential direction, a ring of material was cut from the scaffold,opened into a strip and then clamped at either end of the strip. Thistest was also performed at a rate of 0.5 mm/sec.

(vii) Burst Pressure Testing

The burst pressure for vascular scaffolds was found by monitoringincreasing pressures within the vessel until failure occurred. Apressure catheter was inserted through a cannulating fixture at one endof the vessel. A 60 cc pressure syringe was inserted through a customcannula at the other end of the vessel. The pressure was increased untilrupture, failure or leakage occurred and the pressure change wasrecorded.

(viii) Functionalization of Matrices

To functionalize a matrix, EDC (10 mg) and sulfo-NHS (2 mg) were addedto 5 mL (0.05 mg/mL) of carboxylated quantum dots in aqueous solutionunder gentle stirring for 1 hr at room temperature. EDC activatedheparin (30 mg/20 μl) was prepared according to the same EDC and NHSmethod. In order to conjugate quantum dots and heparin, 5 mg PDA wasadded to the activated quantum dots and heparin solutions under stirringfor 2 hr at room temperature. The quantum dot-heparin (QD-heparin)conjugation can be quenched by adding an equal volume of 1 M Tris buffersolution (pH 7.4) and stored in 4° C. (FIG. 2).

(ix) Encapsulation

Microencapsulation of QD-heparin was performed by double emersion.Briefly, 4 mL of internal aqueous phase containing 30 mg QD-heparin and10 mg bovine serum albumin (BSA) as stabilizer was emulsified in 8 mlsolution of 100 mg PLGA and 100 mg PCL in dichloromethane. The solutionwas emulsified by vortexing for 5 minutes at room temperature. This W/Odispersion was diluted to 200 ml of 1% (w/v) aqueous PVA solution understirring for 4 hr at room temperature. The microcapsules were washedseveral times with deionized water and then lyophilized overnight.

(x) Heparin Release Using IR Irradiation of the Quantum Dot

In order to evaluate the burst release of heparin, 0.55 mg of PLGAmicrocapsules containing QD-heparin were suspended in 2 ml of PBS(phosphate buffered saline). The solution was irradiated for 0, 10 and30 min using an AM1.5 solar simulator at 75 mW/cm2. On days 1, 3 and 5,the samples were then cooled to 4° C., centrifuged at 4500 rpm for 20min and filtered (0.45 μm pore size) to remove any microcapsules for theoptical measurements. Luminescence measurements were performed using anargon ion laser (514.5 nm at 400 mW/cm2) as the excitation source andspectra were collected using a CCD spectrophotometer with an integrationtime of 40 sec.

(xi) Mouse Model

Mice (C57BL6) will be obtained from Jackson laboratories, Bar Harbor Me.All experimentation in mice will be performed aseptically under generalanesthesia (ketamine; 45-75 mg/kg and Xylazine; 10-20 mg/kg, IP). Theincision sites are scrubbed with betadine and wiped with alcohol.Analgesia (Buprenorphine 0.05-0.1 mg/kg, SC) is given post-operativelyafter implantation. Prophylactic antibiotic agents (cefazoline 25 mg/kg,sc) are given to the animals at the time of implantation. The preparedblood vessels (2×0.5 cm) will be implanted in the dorsal subcutaneousspace of mice through a minimal longitudinal midline incision with 2implants per animal. The wound will be closed with interruptedabsorbable sutures and the animals will be sacrificed 1, 2, 4, 8, 12, 18and 24 weeks after implantation for analyses. For the collection ofblood samples, mice will be anesthetized and blood will be retrievedinto heparin containing tubes using cardiac puncture and the mice willbe sacrificed thereafter.

(xii) Sheep Model

A total of 120 sheep will be used. The experimental study will consistof 6 different groups of the blood vessels. Each animal will serve asits own control. Animals will be sacrificed at 1, 3, 6, 12, and 18months after implantation. Animals will be monitored at 0, 1, 2, 3, and4 weeks and monthly for grafts implanted greater than one month.

Sheep will be sedated with Ketamine (5 mg/kg, IM), intubated andanesthetized with Isofluorane (1-3%), and placed on a ventilatoradministering Isoflurane for maintenance. Following Duplex ultrasoundimaging of native femoral arteries the groins will be prepped in asterile fashion and antibiotics administered (cefazolin 25 mg/kg, i.v.).A longitudinal incision will be made overlying the superficial femoralartery, which will then be exposed over a length of 6 to 8 cm. Animalswill receive aspirin for 48 hours prior to surgery (80 mg, p.o.) andheparin will be administered immediately prior to implantation (100U/Kg, i.v.). The femoral artery will then be clamped and dividedproximally and an end-to-side anastomosis created between native andengineered artery with running 7-0 Prolene sutures. The distalanastomosis will then be created in a similar fashion and blood flowrestored through the implant. Duplex ultrasound will then be repeatedusing a sterile intraoperative probe cover to establish arterydimensions and blood flow immediately after implantation. Wounds willthen be closed with absorbable sutures and the animals recovered fromanesthesia using Atropine (0.02 mg/kg i.v.) prior return to standardhousing. Post-operative antibiotics will be administered (Cephazoline 25mg/kg/day) for 3 days following the procedure. Analgesia will beadministered (ketoprofen 2 mg/kg) every 6-12 hours for 3 days. Aspirinwill also be administered (80 mg daily) for 7 days orally foranticoagulation. The animals will be sacrificed 1, 3, 6, 12 and 18months after implantation for analyses. At each time point, 6 animalswill be euthanized for analysis.

Example 2 Electrospun Matrices

An electrospun matrix was formed using the methods outlined inExample 1. A solution of collagen type I, elastin, and PLGA, were used.The collagen type I, elastin, and PLGA were mixed at a relativeconcentration by weight of 45% collagen, 40% PLGA, and 15% elastin.

The resulting fibrous scaffold had a length of 12 cm with a thickness of1 mm. A 2 cm representative sample is depicted in FIG. 3. Thisdemonstrates the feasibility of spinning Type I Collagen and elastininto fibers from nanometer to micrometer diameter using concentrationsfrom 3% to 8% by weight in solution. These results also show that byadding PLGA (MW 110,000) to the mixture, solutions with higher viscosityand improved spinning characteristics could attained. By increasing thesolution concentration to 15%, thicker, stronger scaffolds were able tobe built while maintaining the collagen and elastin components.

Collagen type I stained positively on the decellularized scaffolds,demonstrating uniform distribution. Elastin distribution within thescaffolds was determined by Movat staining. The electrospun scaffoldswith 15% elastin demonstrated a uniform elastin matrix throughout thescaffold wall. These findings indicate that the matrix content anddistribution of the electrospun scaffolds can be manipulated to achievevarious matrix compositions depending on the need.

Results of biocompatibility assays were calculated as a percentage ofnegative control and both electrospun scaffolds performed similarly tothe decellularized blood vessel. These data suggest that thebiocompatibility of electrospun scaffolds is similar to that ofdecellularized scaffold.

Results of mechanical testing for compliance show a typicalpressure-diameter curve for native vessels, as well as fordecellularized and electrospun scaffolds. The diameter change wasapproximately 5% for native vessels and electrospun scaffolds within thephysiologic pressure range which is consistent with the in vivomechanical behavior of porcine and human arteries (FIG. 4). These datademonstrate that the electrospun scaffolds created have a compliancesimilar to that of a native vessel.

Results of the axial and circumferential mechanical tests fromelectrospun scaffolds tended to exhibit a more isotropic behavior.Strain in the axial and circumferential directions were nearlyequivalent before failure occurred.

The results of burst pressure testing show that the burst pressure forthe electrospun construct was 1,425 mmHg or nearly 12 times systolicpressure. These data suggest that electrospun scaffolds have adequateinitial strength and elasticity to withstand the mechanical environmentwhen being surgically placed in the circulatory environment.

Histological analysis of the explanted vascular scaffolds from miceshowed that there was no evidence of inflammation or tissueencapsulation.

Collectively, these results show that it is possible to control thecomposition of electrospun scaffolds for use as vascular grafts. Higherconcentrations of collagen type I and elastin than previously employed,and mixing with PLGA, result in improved spinning characteristics andstrength of grafts, which resist almost 12× systolic pressure. Scaffoldsalso exhibited compliance characteristics similar to native arteries.Scaffolds had an average fiber diameter of 720 nanometers. EDCcrosslinked scaffolds demonstrate superior cell proliferationcharacteristics to glutaraldehyde crosslinked scaffolds as assessed bymitochondrial metabolic activity assay. Cell viability assays did notdemonstrate as pronounced a difference in crosslinking method. Theseresults are some of the first data on biocompatibility of electrospunscaffolds created with biological polymers and PLGA. This workdemonstrates the promise of electrospinning as a fabrication process forvascular graft scaffolds.

Example 3 Cross-Linking of Electrospun Matrices

This example demonstrates how to increase the strength and stability ofthe electrospun scaffold by chemical cross-linking. The scaffolds weresoaked in 20% dextran solution in phosphate buffered saline prior tocrosslinking to reduce hydration-induced swelling and contraction of thescaffold. The scaffolds were crosslinked by immersion in EDC/NHS inMES/EtOH solution for 2 hours at room temperature. Scanning electronmicrographs of the resulting fibers showed fiber diameters of 500 nm orless and a random orientation of fibers. Atomic force microscopy of thescaffold and a confocal image of nanofibers with an adhering endothelialcell demonstrate the scaffold structure. These data show that it ispossible to fabricate vascular scaffolds from biological polymers withmechanics and structure similar to decellularized scaffolds and nativearteries.

Example 4 Distribution of Collagen and Elastin Content

The relative quantity and distribution of collagen and elastin in avascular scaffold is important to the mechanical properties and functionof the seeded graft. The scaffold composition was assessed usinghistochemical analysis for collagen types I, II, and III, elastin andhematoxylin, and eosin (H&E) staining was also performed.

The levels of collagen type I, II, and III, and elastin fordecellularized matrices and collagen type I and elastin for electrospunmatrices were analyzed using computerized histomorphometric analysis.NIH Image/J Image analysis software (National Institutes of Health,Bethesda, Md.) was used for the analysis.

Immunohistochemical analyses using antibodies specific to collagen typesI, II and III were performed on the decellularized and electrospunscaffolds. The decellularized scaffolds showed similar collagen type Iand III in the vascular media, which corresponds to normal bloodvessels. In this study, 45% collagen type I was used to demonstrate thecontrollability of the scaffold fabrication. Collagen type I stainedpositively on the decellularized scaffolds, however, collagen type IIIstained negatively. Elastin distribution within the scaffolds wasdetermined by Movat staining. Abundant elastin fibers were observed inthe entire decellularized scaffold wall with a prominent distribution inthe serosal and luminal surface. The electrospun scaffolds with 15%elastin demonstrated a uniform elastin matrix throughout the scaffoldwall. These findings indicate that decellularized vascular scaffoldspossess matrices similar to normal vessels and that the matrix contentand distribution of the electrospun scaffolds can be manipulated toachieve various matrix compositions depending on the need.

Histograms of the distribution of color were used to determine relativeamounts of each component from each stain against negative controls. Allvalues were normalized by area for comparison. Amounts of collagen I,elastin, and PLGA were known for electrospun matrices because offabrication parameters. Calibrating the image data for relative amountsof collagen utilized both the normalized areas with negative controls,and was calibrated based on known composition of electrospun matrices.

The results demonstrate the composition of collagen I, II, and III, andelastin, in the decellularized scaffolds as well as componentpercentages in electrospun matrices. These studies show that thecollagen and elastin content of decellularized and electrospun scaffoldsis similar to that of native vessels.

Example 5 Compliance Testing of Scaffolds

Compliance mismatch is one of the most common causes of vascular graftfailure, resulting in intimal hyperplasia and occlusion. If the scaffoldis too compliant, it may form an aneurysm. This example describes how totest for compliance of the scaffolds. Decellularized and electrospunvessel shaped scaffolds were immersed in a water bath and cannulated ateither end. One cannula was connected to a column of water and the otherto a drainage tube. The column of water was high enough to create apressure within the vesselshaped scaffold of 120 mmHg. Water was drainedthrough the scaffold in order to lower the pressure in increments of 10mmHg. At each increment, the diameter of the scaffold was recorded usinga digital camera. This process was repeated until the pressure was 0mmHg. Results show the typical pressure-diameter curve for nativevessels, and the experimental curves for decellularized and electrospunscaffolds. The diameter change was approximately 5% for native andelectrospun and 15% for decellularized scaffolds within the physiologicpressure range which is consistent with the in vivo mechanical behaviorof porcine and human arteries. Thus, both decellularized and electrospunscaffolds have a compliance similar to that of a native vessel.

Example 6 Circumferential and Axial Loading of Decellularized andElectrospun Vessels

Vessels must resist higher stress in the circumferential direction thanin the axial direction. Native vessels adapt their mechanics to thisloading environment. It is important that the decellularized andelectrospun scaffolds exhibit a mechanical behavior similar to nativevessels. Thus, mechanical loading tests were performed on thedecellularized vessels and electrospun vessels in the axial andcircumferential directions using a uniaxial load test machine (InstronCorporation, Issaquah, Wash.). An entire vessel-shaped scaffold wasclamped at its cut ends for the axial test. The crosshead speed was setat 0.5 mm/sec and the test was stopped when the strain decreased by 10%after the onset of failure. For testing in the circumferentialdirection, a ring of material was cut from the scaffold, opened into astrip and then clamped at either end of the strip. This test was alsoperformed at a rate of 0.5 mm/sec. Results of the axial andcircumferential mechanical tests from electrospun scaffolds are shown inFIGS. 5A and 5B, respectively.

The electrospun scaffolds tended to exhibit a more isotropic behavior.Strain in the axial and circumferential directions were nearlyequivalent before failure occurred. In general, the decellularizedconstruct exhibits the orthotropic mechanical behavior that is expectedfrom the known mechanical behavior of arteries. In particular, strain inthe circumferential direction is lower than strain in the axialdirection. This was true for scaffolds prior to and after implantation.

The burst pressure for vascular scaffolds was found by monitoringincreasing pressures within the vessel until failure occurred. Apressure catheter was inserted through a cannulating fixture at one endof the vessel. A 60 cc pressure syringe was inserted through a customcannula at the other end of the vessel. The pressure was increased untilfailure or leakage occurred and the pressure change was recorded. Theresults show that the burst pressure for the decellularized constructwas 1,960 mmHg or approximately 16 times systolic pressure. The burstpressure for the electrospun construct was 1,425 mmHg or nearly 12 timessystolic pressure. We demonstrated that both electrospun anddecellularized scaffolds had adequate strength and elasticity and may besubstitutes for native vessels.

Example 7 Isolation, Characterization and Vessel Seeding of SheepProgenitor EPC and MPC

Progenitor EPC and progenitor muscle cells (MPC) were isolated from 60ml peripheral blood of the internal jugular vein of sheep. TheLleukocyte fraction was obtained by centrifuging on a Histopaque densitygradient. Some of the cells were resuspended in medium and plated onfibronectin coated plates. At 24 hr intervals the floating cells weretransferred to new fibronectin coated plates. EPC were induced by growthin EGM-2 medium that contained VEGF and bFGF. The rest of the cells werecultured in the presence of 10 μM 5-Azacytidin for 24 hours. Thereafterfloating cells were transferred to a new fibronectin coated plate andcultured in myogenic medium (DMEM low glucose containing 20% fetalbovine serum, 10% Horse Serum, 1% Chick Embryo extract and 1%antibiotics) in order to induce MPC. EPC and MPC were cultured for 4-6weeks in order to assume differentiated morphology. Immunohistochemicalanalysis of EPC showed that most of the cells expressed VE cadherin andCD31 but not Desmin. However, MPC showed expression of Vimentin andDesmin but not of VE cadherin. The expression of these markers wasmaintained during culture in vitro. These results indicate that culturedEPC and MPC possess EC and muscle cell phenotype, respectively.

EPC were labeled by PKH 26 green fluorescent dye and MPC were labeled byPKH 27 red fluorescent dye. Labeled EPC and MPC were seeded on theluminal and the outer surfaces of decellularized vessel segments,respectively, in order to demonstrate the biocompatibility of thedecellularized vessel. After 7 days the presence of red and greenlabeled cells on the decellularized vessel was noted. In addition,seeded vessels were seeded with a suspension of red-labeled MPC andgreen labeled-EPC (5×10⁶ cells/ml) and cells were allowed to grow for 7days. The vessels were embedded in OCT media in order to obtain frozensections. The sections were stained with DAPI. To detect cell nuclei,sections were visualized using a fluorescent microscope. Data shows thatEPC were maintained on the luminal side of the scaffold and MPC on theserosal surface.

Example 8 Cell Attachment

A confluent monolayer of endothelial cells is the most important barrieragainst thrombosis formation. Endothelial cell mediated NO production isimportant in maintaining the vascular tone. To examine cell attachment,the decellularized and electrospun vessels were seeded with endothelialcells. Cell attachment was assessed using scanning electron microscopyof scaffolds seeded with a mouse endothelial cell line (MS1). SEMmicrographs reveal a confluent monolayer on the inner surface of boththe decellularized and electrospun vessels at 48 hours. These resultsindicate that endothelial cells form confluent monolayers ondecellularized and electrospun scaffolds.

Example 9 Biocompatibility (Cell Viability and Proliferation)

Long-term viability of cells is necessary for the seeded scaffold toremodel itself into a viable, patent vessel. To test for cell viability,decellularized and electrospun constructs were placed in 24-well plateswith approximately 100 mg of material per well. Four different types ofmaterial were tested for biocompatibility and cell survival, with onenegative control well with no material: (1) GA-NFS (1% glutaraldehydecrosslinked electrospun scaffold); (2) EDC-NFS (EDC-crosslinkedelectrospun scaffold); (3) nBV (natural blood vessel, decellularized);(4) Latex (latex rubber, positive control).

Endothelial cells were seeded in the wells on a scaffold for testing viathe direct contact method. For cell viability, cell layers were rinsedwith PBS. 0.005% (w/v) neutral red was added in culture medium. Theneutral red solution was removed after 4 hours incubation at 37° C. with1% acetic acid and 50% ethanol solution by volume was added for dyeextraction, and dye extraction was shaken for 5 minutes. Absorbance wasthen measured at 540 nm using a spectrophotometer. The intensity of redcolor obtained was directly proportional to the viability of the cellsand inversely proportional to the toxicity of the material. Results werereported as a percentage of negative control, and both electrospunscaffolds performed similarly to the decellularized blood vessel (FIG.6A).

Cell proliferation was tested using the mitochondrial metabolic activityassay. Cell layers were first rinsed with PBS. MTT solution was added at1 mg/mL in PBS containing 1 mg/mL glucose. MTT solution was removedafter 4 hours incubation at 37° C. Dimethyl sulfoxide (DMSO) was used todissolve insoluble formazan crystals, and the absorbance at 540spectrophotometer. The intensity of blue color was directly proportionalto the metabolic activity of the cell populations and inverselyproportional to the toxicity of the material or extract. Thegluataraldehyde treated matrices show more pronounced differences thanin proliferation assays, with EDC treated scaffolds being similar tonatural blood vessels (FIG. 6B).

Cell viability and proliferation testing was also performed to determinethe effects of various concentrations of gadolinium (Gd) on thescaffolds, on cell survival (FIG. 7). The tests revealed little effectof Gd levels on cell viability or survival. The results indicate thatboth scaffolds can promote cell growth and thus may be used for thebioengineering of vascular grafts.

Example 10 External Functionalization of Matrices

This example describes how to generate matrices with image enhancingagents and quantum dots. In particular, Gd-DPTA and quantum dotfunctionalization of an external scaffold. The scaffold can be anybiocompatible substrate, such as a synthetic PGA matrix, an electrospunmatrix, or a decellularized matrix. At present, no clinically availablevascular graft allows for noninvasive monitoring of the integration ofthe graft in vivo, nor does any graft incorporate anticoagulants intoits structure. A reliable method is needed to attach nanomaterials toscaffolds, e.g., vascular scaffolds, in order to increase functionality,in particular as a material marker and for anticoagulation. CarboxylatedGd and quantum dot (QD) materials were coupled to the surface of boththe decellularized and the electrospun scaffolds using an EDC/sulfo-NHSmethod. Any unreacted material was quenched and removed by rinsing thescaffold with 0.1 M Tris buffer. The liquid from the final washing wascolorless under UV elimination.

Under blacklight illumination the functionalized scaffold showsmulticolor fluorescence. Areas of red-orange emission are from thequantum dots. The pale white color, which is stronger in intensity thanthe control tissue, comes from the Gd containing material that canfluoresce with a pale blue color. The data shows that it is possible toincorporate heparin onto the surface of a scaffold. The scaffolds arealso able to bind Gd.

Example 11 Internal Functionalization of Matrices

This example describes the production of electrospun matrices with imageenhancing agents and therapeutic agents. In particular, Gd-DPTA and QDaddition to the internal electrospun scaffolds. Fabricating vascularscaffolds using electrospinning provides an opportunity to incorporateimage enhancing agents within the bulk material. Solutions were spunsuccessfully containing gadolinium diethylenetriamine pentacetic acid(Gd-DPTA) in HFP at a concentration of 15 mg/mL and with quantum dotsadded at a concentration of 8% by volume from a quantum dot solution of25.5 nmol/mL in toluene. No morphological change was noted in thescaffolds due to the addition of the Gd-DPTA or the QDs. These resultsshow that incorporating nanoparticles into the scaffolds has only aminimal effect on the morphology of the resulting structure.

Example 12 Matrices with Quantum Dots

This example describes how to couple therapeutic agents, such as heparinto the quantum dots (QD). Heparin is a potent anticoagulant agent. Toavoid systemic administration, a method is needed to control the releaseof heparin from the vascular scaffold and to bind the heparin to thescaffold. In this experiment, EDC (10 mg) and sulfo-NHS (2 mg) was addedinto the 5 mL (0.05 mg/mL) of carboxylated quantum dots in aqueoussolution under gentle stirring for 1 hr at room temperature. EDCactivated heparin (30 mg) was prepared according to the same EDC and NHSmethod as described above. In order to conjugate quantum dots andheparin, 5 mg phenylene diamine (PDA) was added to the activated quantumdots and heparin solutions while stirring for 2 hr at room temperature.The quantum dot-heparin (QD-heparin) conjugation can be quenched byadding an equal volume of 1 M Tris buffer solution (pH 7.4) and storedin 4° C.

Microencapsulation of QD-heparin was performed by double emersion.Briefly, 4 mL of internal aqueous phase containing 30 mg QD-heparinconjugation and 10 mg bovine serum albumin emulsified in 8 mL of asolution of 100 mg PLGA (MW; 110,000) and 100 mg PCL (MW; 110,000) inDCM. The solution was emulsified by vortexing for 5 min at roomtemperature. This W/O dispersion was diluted into 200 mL of 1% (w/v)aqueous PVA solution under stirring for 4 hr at room temperature. Themicrocapsules (MCs) were washed several times with deionized water andthen lyophilized overnight. QD-heparin nanocapsules (NC) wereincorporated into scaffolds by placing the functionalized vascularscaffold in 1 wt % PLL in PBS. Vascular scaffolds were immersed in thePLL-nanocapsule solution for 3-4 hours, and lyophilized beforesterilization with gamma irradiation.

A fluorescence image of an isolated microcapsule containing quantum dotsshows that the characteristic fluorescence from the quantum dots used inthis experiment is at 500 nm. The data show that it is possible to bindheparin to quantum dots and encapsulate the bound heparin in abiodegradable polymer, for attachment to the vascular scaffold.

Example 13 Release Kinetics of Heparin: In Vitro Release of Heparin andBurst Release by Irradiation

To assess the effectiveness of quantum dots for controlled delivery ofheparin, the release kinetics of the drug was analyzed following anirradiation burst. In order to evaluate the burst release of heparin,0.55 mg of PLGA microcapsules with QD-heparin were suspended in 2 ml ofbuffered saline solution. The solutions were irradiated for 0.0, 10, and30 min using an AM1.5 solar simulator at 75 mW/cm². On days 1, 3, and 5the samples were then cooled to 4° C. and centrifuged at 4500 rpm for 20min. The solutions were filtered (0.45 m pore size) to remove anymicrocapsules for the optical measurements.

Luminescence measurements were performed using an argon ion laser (514.5nm at 400 mW/cm²) as the excitation source and spectra were collectedusing a CCD spectrophotometer with an integration time of 40 sec.Irradiated samples showed increased luminescence over time indicating a“burst effect”. The kinetic profile of heparin confirms that irradiationinduced the burst release out of functionalized microcapsules. Heparinrelease was monitored by optical analysis (FIG. 8A) and biochemicalanalysis (FIG. 8B). These results indicate that NIR can be used toinitiate the release of heparin from the QD-heparin microcapsules.

Normally, heparin is administered at the site of implantationimmediately following surgery to prevent acute thrombosis. Afterwards,heparin is administrated within the first week twice a day by injection.In order to improve the patient's compliance, heparin could beimmobilized in vascular scaffolds for extended period of time. However,the immobilization of heparin to the scaffolds results in a slow releaseof heparin which is not appropriate for thrombus prevention. Toaccelerate the burst release of heparin, near infrared (NIR) irradiationof the quantum dots bound to heparin can to be used to achieve thisgoal.

Example 14 Determination of the Remaining Heparin in Retrieved VesselImplants from Mice

To assess the effectiveness of heparin in an in vivo model, heparin mustbe evaluated after the explantation of the scaffold. The remainingheparin in functionalized blood vessels (heparin-QD) implanted in micewas determined by toluidine blue staining and most of the heparin isshown to have diffused out of the vessel two weeks after implantation.The heparin content was analyzed by a Rotachrome kit and the dataconfirms that very little heparin remains after two weeks. The data showthat the activity of heparin was successfully prolonged in the scaffoldbeyond its normal 1-2 hour half-life (FIG. 9).

The inflammatory response of quantum dots should be addressed forclinical applications. From the histological analysis of the explantedvascular scaffolds from mice, there was no evidence of inflammation ortissue encapsulation. The data indicate that conjugated heparin had onlya minimal inflammatory response.

Example 15 Evaluation of the Anti-Thrombogenic Properties of HeparinImmobilized Vessels

Although heparin is a powerful anticoagulant, it was important to verifythat this property still exists after immobilization. Two methods ofheparin binding were tested. Thirty milligrams of heparin was incubatedin 20 mM EDC and 10 mM sulfo-NHS in PBS for 2 hours at room temperature,and a 3 mm diameter decellularized scaffold was then immersed inheparin-EDC solution for 2 hours at room temperature. Aftercrosslinking, the sample was rinsed in PBS several times to completelyremove residual EDC. Subsequently immobilization of heparin by physicaladsorption was performed using Poly(L-lysine) (PLL): The 3 mm diameterdecellularized scaffold was incubated in 2 mg/mL PLL solution for 2hours at room temperature. The PLL-adsorbed scaffold was immersed in 15mg/mL heparin solution for 1 hour at room temperature. Theanti-thrombogenic property of each method was evaluated using wholeblood from sheep by toluidine blue staining. Immediate coagulation wasobserved from the decellularized scaffold while no significant sign ofcoagulation was found from both EDC and PLL reacted decellularizedscaffolds 36 hours after blood treatment. The heparin-PLL decellularizedscaffold demonstrated the weakest staining which indicated the highestloading of heparin in the scaffold. These results showed thatimmobilized heparin was effective in preventing thrombus.

Example 16 Enhanced MRI Imaging

This example demonstrates the improved imaging observed with gadolinium.In vitro experiments were conducted on cell scaffolds with gadolinium todetermine the improvement in magnetic resonance imaging. Cylindricalcell scaffolds 20 millimeters long with an internal radius of 10millimeters and a outer radius of 14 millimeters were created withdifferent Gd loading concentrations. Cell scaffolds were individuallyplaced in test tubes and submerged in PBS. The four test tubes werearranged left to right in the following order: non-functionalized cellscaffold (control 1), functionalized scaffold (control 2), 1×Gdconcentration cell scaffold, 100×Gd concentration (control 3) and a1000×Gd concentration cell scaffold. (100× designates a concentration insolution during functionalization of 55 mg/kg of Gd-DPTA) Axial T1weighted spin echo images were acquired on a on a GE HealthcareTechnologies magnetic resonance imaging (MRI) 1.5T TwinSpeed scanner.

The T1 weighted image acquired with a phased array coil and a 200millisecond repetition time (TR) was obtained. Additional imagingparameters are as follows: echo time (TE)=13 ms, slice thickness=0.8 mm,256×128, field of view (FOV)=12 cm×6 cm, number of averages=100, andphase direction was right to left. The cell scaffolding loaded with1000×Gd (right most test tube) is clearly visible compared to controls1, 2, and 3. Samples were washed twice with TRIS buffer and PBS andstored in PBS for 2 weeks prior to imaging.

The previously described experiment was repeated for two different Gdloaded scaffold preparations: surface and volume loading. The scaffoldon the left is a cylindrically shaped scaffold identical to thepreviously described experiment with a surface loaded 1000×Gdpreparation. The scaffold on the right is a planar sheet of scaffoldwith the Gd embedded throughout the electrospun fibers as describedpreviously. The scaffold that had the Gd electrospun into the fibershowed a much higher contrast. The normalized signal intensities of thescaffold for the surface preparation and volume preparation are 1.5±0.2and 2.98±0.35, respectively. The data on MRI Imaging of Gd loadedscaffolds showed that Gd increases MRI contrast in proportion to thelevel of Gd loaded in the scaffold.

Example 17 In Vivo Preliminary Data on Rodents

Although Gd may be maintained in the scaffolds in vitro, it is necessaryto demonstrate that it retains functionality in vivo. This experimentinvestigates the in vivo functionality of the scaffolds. Electrospunvascular scaffolds were implanted subcutaneously in a mouse for twoweeks prior to imaging. Gd was added to one of the vascular scaffolds toenhance its contrast on a T1 weighted image. A sagittal localizer imagewas acquired from the mouse and a T1 weighted coronal image containingthe two scaffolds was prescribed off the sagittal image. The importantimaging parameters of the T1 weighted image are repetition time (TR) 300milliseconds, echo time (TE) 14 milliseconds, and slice thickness 2millimeters. A 50% improvement in image contrast of the Gd scaffoldcompared to the control. These results in a rodent model demonstratethat the characteristics seen in vitro are maintained in vivo.

Example 18 Ex Vivo Preliminary Data on Sheep Engineered Vessels

In vivo results in the rodent model were limited to subcutaneousspecimens. It was necessary to demonstrate similar results in a scaffoldexposed to blood flow in a large animal model. To determine thefeasibility of using the cell seeded scaffolds containing thenanoparticles (heparin conjugated with quantum dots and Gd-DTPA),femoral artery bypass procedures were performed in sheep. Peripheralblood samples were collected, circulating progenitor cells were selectedand differentiated into endothelial and smooth muscle cells in culture.Each cell type was grown, expanded separately and seeded ondecellularized vascular scaffolds containing the nanoparticles (30 mmlong). Nanoparticle containing scaffolds without cells served as acontrol. Under general anesthesia, sheep femoral arteries were imagedwith duplex ultrasonography (B-mode ultrasound and Doppler spectralanalysis) with a high resolution 15 MHz probe (HDI-5000, ATL) prior toscaffold implantation. The femoral artery was exposed through alongitudinal incision over a length of 6 to 8 cm. Aspirin and heparinwere used as anticoagulation and the femoral artery was clamped anddivided proximally. An end-to-side anastomosis was created betweennative and engineered artery. The distal anastomosis was created in asimilar fashion and blood flow restored through the implant followed byligation of native femoral artery between the two anastomoses. Dopplerultrasonography was performed using a sterile probe to establishscaffold dimensions and blood flow after implantation. Wounds wereclosed and the animals recovered from anesthesia prior to 3500 return tostandard housing. Aspirin was administered routinely for 7 days orallyfor anticoagulation.

Duplex ultrasound imaging was performed to determine the presence ofthrombosis, lumen narrowing intimal hyperplasia and graft wallstricture, and graft aneurismal degeneration. Longitudinal andcross-sectional images of the pre- and post operative arterial segmentsshowed a patent lumen 0 with similar peak systolic, end-diastolic andtime averaged velocities as the normal artery. The arterial wallthickness and luminal diameter of the engineered bypass was similar tonative artery. The engineered arterial bypass and the contralateralnormal femoral artery were scanned with MRI. T1 weighted spin echo MRimages were acquired with the following parameters: 256×126 matrix, 12×6mm FOV, 400 ms TR, 13 ms TE, 1 mm slice thickness, and 50 excitations.Average signal intensities of the samples were normalized by thebackground water intensity to account for receiver coil nonuniformities.The normalized intensities were 2.62 and 2.10 for the scaffold andnormal vessel, respectively.

This experiment was repeated at several different TRs and the signalintensity measured for the scaffold and the normal vessels. As expected,the signal intensity for the gadolinium enhanced scaffold is alwaysgreater than the normal vessel. These results confirmed that Gd andheparin loaded decellularized scaffolds maintain patency in a sheepmodel and maintain MRI contrast.

Gadolinium is a MR contrast agent that enhances images primarily bydecreasing the spin-lattice relaxation time (T1) of protons in tissues.Unlike radionuclides, it will remain effective as long as it islocalized in the engineered vessel. These results shown in vitro throughrepeated rinsing of the Gd doped scaffolds and in vivo through imagingof the engineered vessel, that the functionalized Gd nanoparticles arestable in the matrix. Within the first 3 months, approximately 80% ofthe graft will be remodeled. The Gd localized in the matrix willinitially enhance the imaging of the graft. The change in MR signal overtime, as the concentration of Gd decreases with remodeling of thevascular graft, will allow us to quantify the remodeling rates.

Example 19 Histomorphological Characteristics of Bypass Grafts in Sheep

To demonstrate cell attachment on the retrieved engineered vesselsinitially seeded with endothelial and muscle cells, scanning electronmicroscopy was performed 2 weeks after implantation. The implanteddecellularized scaffolds seeded with cells showed a uniform cellattachment on the luminal surface of the engineered artery similar tonormal vessels. The scaffolds without cells failed to exhibit cellattachment. These observations indicate that the cells seeded ondecellularized vascular scaffolds are able to survive and remainattached after surgery.

To assess the histo-morphological characteristics of the retrievedtissue from engineered arterial bypass grafts in sheep, histologicalevaluation was performed. The engineered arterial specimens were fixed,processed and stained with hematoxylin and eosin (H&E) and Movatstaining. The cell seeded engineered grafts contained uniformcellularity throughout the vascular walls. Abundant elastin fibers wereobserved in the entire arterial wall with a prominent distribution inthe serosa and luminal surface. These findings demonstrate that theengineered vessels, seeded with peripheral blood derived progenitorcells differentiated into endothelial and smooth muscle cells, are ableto show an adequate cellular architecture similar to native vessels.

Collectively, these studies show that it is possible to fabricate andfunctionalize both decellularized and electrospun scaffolds with cells(endothelial and smooth muscle) and nanomaterials (quantumdot—conjugated heparin) that are known to have a positive therapeuticbenefit. Moreover, the data shows the successful incorporation ofmolecules (gadolinium) enhancing MRI contrast to monitor the engineeredvessels over time. The combination of functionalization and imagingoffers the potential for making these scaffolds an ideal vascularsubstitute. The matrices are biocompatible, possess the ideal physicaland structural properties, and have been shown to be functional for over4 months in the carotid artery of sheep.

Example 20 To Characterize the Engineered Vascular Grafts

In order for a vessel to function normally, it should have theappropriate structural properties to accommodate intermittent volumechanges. In pathologic conditions, normal vessel function and mechanicalproperties may be compromised. To translate the use of bioengineeredvessels to patients, it is first necessary to confirm that normalvessels are being formed, and that they retain adequate phenotypic andfunctional characteristics over time, especially with growth.

(i) Mechanical Testing

Understanding the mechanical properties of explanted vessels providesinformation about the adaptive remodeling those vessels have undergonewhile in the host animal. Mechanical testing will include arterialelongation (axial and circumferential), compliance, burst pressure,stress relaxation, and creep.

(ii) Phenotypic and Composition Analyses

Histological and immunohistochemical analysis can be performed on theretrieved vascular grafts. Longitudinal and cross sections will be takenfrom the transition zones between native vessels and graft and from therest of the graft. Specimens will be fixed, processed and stained withHematoxylin and eosin (H&E) and Masson's trichrome. Cross-sectionalareas of the adventitia, media, intima and lumen will be measured usingcomputer-assisted analysis of digital images (NIH Image Software). Inaddition to cross-sectional analysis of the engineered artery body, aseparate analysis will be performed for the anastomoses region betweennative and engineered arteries. The proximal and distal anastomoses willbe fixed in formalin, embedded in paraffin, and then cut incross-section for analysis of lumen caliber and artery wall thickeningin step-sections spanning each anastomosis. In parallel, quantitation ofthrombus formation will be performed using H&E staining. The phenotypiccharacteristics of the retrieved tissues will be determined over time.

To determine the degree of endothelial and smooth muscle content of thebioengineered vessels over time, in comparison to normal tissues,multiple molecular markers will be probed immunocytochemically and withWestern blot analyses, as described above. These markers will includeAnti-Desmin and Anti-Alpha Smooth Muscle Actin, which specificallydetects smooth muscle cells. Endothelialization will be evaluated byanti-von-Willebrand factor anti-CD-31 and anti-VEGF receptor, KDR,antibodies, which stain EC specifically. Cell proliferation andapoptosis in engineered arteries will be determined by BrdUincorporation and TUNEL staining.

The composition and distribution of extracellular matrix components,such as collagen and elastin, are important for the normal function ofblood vessels. While the collagen network is responsible for tensilestrength, elastin is important for the elastic recovery of the vessel.Therefore, an assessment of the collagen and elastin content anddistribution of the retrieved tissues over time will be performed withhistological and quantitative biochemical assays. To determine whetherthe retrieved vessels possess normal concentrations of collagen andelastin, as compared to normal controls, the total collagen and elastincontent per unit wet weight of the retrieved tissue samples will bemeasured quantitatively using the Sircol collagen and the Fastin elastinassay systems (Accurate Chemical & Scientific Corporation, Westbury,N.Y.). To determine the anatomical distribution of collagen within theengineered vessels, as compared to controls, Immunocytochemicallocalization of collagen types I, II and III will be performed usingspecific monoclonal antibodies (Southern Biotechnology Associates, Inc.,Birmingham, Ala.) and with the elastin-specific stain, Movat.

(iii). Physiological Analysis

The ability to synthesize vasoactive agents such as Nitric Oxide (NO)will further determine the functionality of the engineered vascularscaffolds. There is increasing evidence on the importance of NO invascular hemostasis. NO contributes to resting vascular tone, impairsplatelet activation, and prevents leukocyte adhesion to the endothelium.

Briefly, guinea pig thoracic aorta will be harvested, the endotheliumlayer removed by gentle rubbing and cut into 5-mm segments. Each segmentwill be suspended between 2 tungsten stirrups for measurement ofisometric tension. The vessel segments placed in an organ chamber with10 ml Kreb's buffer solution at 37° C. with a mixture of 5% CO₂, 15% O₂and a balance of N2. Each vessel (2-3 cm in length) is tied to a 21 Gneedle, which was attached to plastic IV tubing and placed above theorgan chamber with the fresh aortic segment. The segments will becontracted with 80 mM KCl Kreb's buffer in a stepwise fashion to obtaina resting tension of 4 g. After resting for 90 minutes, the segments arecontracted in response to prostaglandin F2α up to a final concentrationof 10⁻⁷M and until a stable contraction of approximately 50% of maximumKCl-induced contraction achieved. Vasoactive agents and antagonists arethen added using an infusion pump through the vessels to induce NOproduction. Doses of the vasoactive agents between 10⁻⁷-10⁻³ M will betested and dose-response curves will be constructed.

Example 21 Preparation of Decellularized Tissues and Organs

The following method describes a process for removing the entirecellular content of an organ or tissue without destroying the complexthree-dimensional infra-structure of the organ or tissue.

(i) Organs

A liver was surgically removed from a C7 black mouse using standardtechniques for tissue removal. The liver was placed in a flaskcontaining a suitable volume of distilled water to cover the isolatedliver. A magnetic stir plate and magnetic stirrer were used to rotatethe isolated liver in the distilled water at a suitable speed for 24-48hours at 4° C. This process removes the cellular debris and cellmembrane surrounding the isolated liver.

After this first removal step, the distilled water was replaced with a0.05% ammonium hydroxide solution containing 0.5% Triton X-100. Theliver was rotated in this solution for 72 hours at 4° C. using amagnetic stir plate and magnetic stirrer. This alkaline solutionsolubilized the nuclear and cytoplasmic components of the isolatedliver. The detergent Triton X-100, was used to remove the nuclearcomponents of the liver, while the ammonium hydroxide solution was usedto lyse the cell membrane and cytoplasmic proteins of the isolatedliver.

The isolated liver was then washed with distilled water for 2448 hoursat 4° C. using a magnetic stir plate and magnetic stirrer. After thiswashing step, removal of cellular components from the isolated wasconfirmed by histological analysis of a small piece of the liver. Ifnecessary, the isolated kidney was again treated with the ammoniumhydroxide solution containing Triton X-100 until the entire cellularcontent of the isolated liver was removed. After removal of thesolubilized components, a collagenous three-dimensional framework in theshape of the isolated liver was produced.

This decellularized liver was equilibrated with 1× phosphate buffersolution (PBS) by rotating the decellularized liver overnight at 4° C.using a magnetic stir plate and magnetic stirrer. After equilibration,the decellularized liver was lyophilized overnight under vacuum. Thelyophilized liver was sterilized for 72 hours using ethylene oxide gas.After sterilization, the decellularized liver was either usedimmediately, or stored at 4° C. or at room temperature until required.Stored organs were equilibrated in the tissue culture medium overnightat 4° C. prior to seeding with cultured cells.

(ii) Blood Vessels

Porcine arterial segments were obtained from pigs (20 to 30 Kg, PatersonFarm, Mass.). Blood vessels with an internal luminal size of 3 to 4 mmwithout branches were retrieved 6 to 8 cm below the bifurcation of thecarotid arteries and cut into segments of approximately 4 cm in length.Vessels were placed in distilled water for one hour to induce red bloodcell lysis, thoroughly washed, and incubated in a decellularizationsolution containing 1% Triton 100X and 0.1% ammonium hydroxide in salinefor 48 hours in a mechanical rotating shaker (120 RPM) at 4° C. Thisprocess was repeated twice and followed by extensive washes in distilledwater. The decellularized vessels were placed in PBS for 24 hours andthen lyophilized (Virtis; Gardiner, N.Y.) and sterilized in cold gas.The decellular vessels were analyzed by Hematoxylin and Eosin.

The results of the decellularization process showed that arterialsegments that underwent decellularization and lyophilization maintainedtheir tubular appearance and did not shrink significantly. Hematoxylinand eosin (H&E) staining of decellularized vessels showed layers ofcollagenous fibers within the vessel walls, suggesting that thedecellularization process did not damage the non-cellular components ofthe native vessel wall.

To examine the composition of the decellularized vessel wall matrix,Movat staining was performed using Russell-Movat pentachrome stain kitfrom American Master Tech Scientific Inc. (Lodi, Calif.), according tothe manufacturer's instructions. Movat staining was performed thatdistinguishes between different extracellular components. Movat stainingresults in black staining of elastin fibers, yellow staining ofcollagen, blue to green staining of proteoglycans, red staining ofmuscle and dark red staining for fibrous tissue. Surface andcross-sectional ultrastructure of native and decellularized vessels wereexamined by scanning electron microscopy (SEM; Model S-2260N, HitachiCo. Ltd., Japan). Samples were observed under an environmental SEM whichallows morphological analysis of electrically non conductive sampleswithout metallic coating and by maintaining low vacuum mode in samplechamber. Parameters such as accelerating voltage (from 15 to 30 kVolts),beam intensity and vapor pressure were tuned for each individual samplein order to obtain sharp images and detailed morphological information.SEM examination of the luminal side of decellularized vessels showed theremoval of the native cell layer, leaving a smooth surface. Crosssection of the vessel wall revealed many collagen layers that are denserin the luminal side.

The mechanical behavior of decellularized vessels was also assessed byexamining how the decellularized vessels responded to mechanical loadingby measuring compliance, mechanical loading resistance in the axial andcircumferential directions, and burst strength. Mechanical testing wascarried out at room temperature with an Instron material testing system(model 5544; Norwood, Mass.), equipped with a 1000 Newton load cell.Tissue specimens of native porcine artery and decellular vessels werecut into strips of 4 cm×1 cm for axial testing, and 1 cm×1 cm forcircumferential testing from a total of six specimens. The specimenswere stored in distilled water at room temperature for up to 4 hoursprior to testing. Before loading the specimens, the ends of each segmentwere wrapped with double sided sandpaper to minimize any motion betweenthe specimen and the grips. Samples were mounted under zero stressconditions. No preconditioning of the samples was performed. Thecrosshead speed was 2.5 mm/min. The system measured the stress-strainbehavior until rupture occurred. Averages and standard deviations (SD)of the stress applied were calculated and plotted against theproportional change in size (strain). Burst strength of the vesselsmeasured by increasing the pressure in the cannulated vessels untilrupture occurred.

Results showed the typical pressure-diameter curve for a pure collagenvessel (FIG. 10A). The diameter change ranged from 4-5% for both vesselswhich is consistent with the in vivo mechanical behavior of largearteries in humans. Mechanical loading tests were performed on thedecellularized vessels in the axial and circumferential directions usinga uniaxial load test machine. The decellularized vessels exhibited theorthotropic mechanical behavior that is expected from the knownmechanical behavior of arteries. In particular, strain in thecircumferential direction occurs to a lesser extent than strain in theaxial direction (FIG. 10B). The burst pressure for the de-cellularizedconstruct was 1,960 mmHg or approximately 16 times systolic pressure.Taken together, these results indicate that the decellularization ofporcine arterial segments reliably remove cellular components from thevessels while preserving the extracellular matrix within the vessel walland its mechanical strength.

Example 22 Isolation of Human Saphenous Endothelial Cells (HSVEC)

After obtaining an informed consent, discarded segments of the saphenousvein were obtained from patients undergoing CABG. The vessels wereplaced in M199 medium containing 20% FBS. Segments of approximately 1 cmwere clamped at both ends and filled with 1 mg/ml collagenase type II inserum-free M199 medium and placed in a 5% CO₂ humidified incubator for20 minutes at 37° C. The vein was then flushed with 50 ml of Ml 99 andcells were collected by centrifugation for 10 minutes at 300×g.Subsequently, cells were resuspended in 3 ml EGM-2 medium containing 20%FBS and seeded in 35 mm (radius) gelatin-coated (0.2% PBS) dishes. Afterreaching near confluence, HSVEC were purified by immunoisolation usingUlex europeaus I lectin coated magnetic beads (Dynal, Norway), asdescribed in Jackson, et al., (1990) J. Cell Sci.: 257-262.Subsequently, HSVEC were cultured in gelatin-coated 100 mm (radius)gelatin-coated dishes and the medium was replaced every three days. Thecell harvesting and immunoisolation processes were repeated with severalsamples from different patients with similar results and the cells werecultured for more than 12 passages. For immunohistochemical analyses,HSVEC seeded onto 8 chamber slide were fixed with 2% paraformaldehyde,washed and incubated with primary antibodies. Biotinylated secondaryantibodies were used and were detected by FITC-conjugated avidin. Theslides were mounted with DAPI-containing media and visualized underfluorescent microscopy. Cultured HSVEC in 10 cm dishes were dissolved inbuffer containing 10 mM Tris, pH 7.0, 50 mM NaCl, 1% Triton and proteaseinhibitors. Immunoprecipitations and Western blots were performed withanti-KDR antibodies.

Example 23 Bioreactor Chamber

To produce blood vessels that have mechanical properties that mimicnative in vivo blood vessel, i.e., are able to withstand variations inblood flow and pulse rate, a bioreactor chamber was designed and used toprecondition the engineered blood vessels.

The fabrication and enhancement of physical properties of a tissueengineered small-caliber blood vessels (TEBV) is illustrated usingdecellularized porcine carotid arteries that are coated with humanendothelial cells. The cell seeded matrix is placed in bioreactor wherethey were incubated in physiological flow and pressure conditions for 1week.

The method involves attaching the sterile decellularized blood to thebioreactor via luer fittings (FIG. 12). Human endothelial cells at adensity of 1.5×10⁶ cells/ml were inserted and sealed into the lumen ofthe vessel to allow for static seeding. The bioreactor was then rotated90 degrees every 3 minutes for two revolutions. After rotations, thecells were incubated for an additional 30 minutes statically prior topositioning the bioreactor (vessel) in the flow system to induce shearstress. Steady flow was increased steadily over 4 days to induce wallshear stress values ranging from 3 to 20 dynes/cm². Pulsatile flowconditions followed for two additional days generating wall shear stressvalues ranging from 10 to 25 dynes/cm² and a pressure distribution from80 to 180 mmHg. Cryostat sections (8 μm) were analyzed histologicallywith hematoxylin and eosin (H&E) to discern endothelial cells binding.

A functional confluent EC layer is an essential component for theprevention of graft thrombosis and atherosclerosis. Therefore, it isimportant to have proper seeding of the luminal in the decellularizedblood vessel. Using the bioreactor, endothelial cells were found to beprovide a uniform confluent layer of endothelium.

The mechanical properties (burst pressure, stress, and strain) of thedecellularized vessels were very similar to the native human artery.Hematoxylin and Eosin staining (H&E) illustrated that endothelial cells,seeded on the luminal side, adhered to the matrix and formed a uniformmonolayer. These results reveal that, TEBV coated with endothelial cellsposses many of the morphologic and functional characteristics ofsmall-caliber vessels. TEBV may potentially be useful clinically asvascular grafts.

Example 24 Seeding Decellularized Porcine Arterial Segments with HSVEC

One of the vessel outlets from the bioreactor of described in Example 23was sealed and approximately 0.5 ml of HSVEC (5×10⁶ cells/ml) suspensionwas inserted into the other outlet and left inside the vessel for anhour with rotations every 15 minutes. Thereafter EBM-2 was gently addedand the medium was replaced every 2-3 days. Cells were allowed to grow 5to 7 days on the matrix. Sixteen HSVEC seeded decellularized porcinevessels were implanted in the subcutaneous space of 8 athymic mice andretrieved for histological analysis after 7 days. The retrieved matriceswere immersed in O.C.T. compound (Sakura Finetek; Torrance, Calif.) andfrozen in liquid nitrogen. Cryostat sections (5 μm) were analyzedhistologically with hematoxylin and eosin (H&E) and by immunostainingwith anti CD-31 antibodies. Control staining was performed using normalrabbit serum. The primary antibodies were detected using biotinylatedsecondary antibodies and the avidin-biotin-immunoperoxidase method. Thereaction was developed using diaminobenzidin (DAB) solution (VectorLaboratories, Burlingame, Calif.). As a control, the primary antibodywas replaced with normal goat serum.

Although the decellularized arterial segments maintained an intacttubular structure, allowing them to serve as a short-term conduit, afunctional confluent EC layer is an essential component for theprevention of graft thrombosis and atherosclerosis. Accordingly, theclinically relevant source of human EC was examined. EC were isolatedfrom discarded human saphenous vein segments (HSVEC) and purified fromthe primary culture by immunoisolation, using Ulex europeaus I lectin.HSVEC from several donors were successfully expanded in culture, longterm, and they formed a typical EC monolayer at confluence.Immunohistochemical analysis of HSVEC with anti-vonWillebrand factor(vWF) and anti-KDR antibodies showed typical punctuated staining.Anti-CD31 staining showed a specific membranal staining. In contrast,the HSVEC were negative for smooth muscle cell (SMC) actin staining. Theexpression of an endothelial-specific VEGF receptor 2 (KDR) wasconfirmed by immuno-precipitation and Western blot with anti-KDRantibodies. The expression of these EC markers was maintained duringculture in vitro. These results indicate that cultured HSVEC possess ECphenotype and express specific EC genes and functional growth factorreceptors.

HSVEC were seeded on the lumen of decellularized porcine vessels and thecell seeded vessels were incubated in culture for additional 5-7 days.SEM examination of seeded vessels showed surface coverage with a uniformlayer of cells. Segments of seeded vessels were further processed formRNA isolation. RT-PCR analysis of cell-seeded decellularized vesselsrevealed expression of VEGF receptors, Flt-1, KDR and NRP-1 as well asGAPDH, similar to the expression levels in cultured HSVEC. In contrast,the expression of these genes could not be detected in the unseededdecellularized vessels, confirming a complete decellularization of thearterial segments. These results confirmed the presence of HSVEC on thedecellularized vessels in vitro 5-7 days after cell seeding.

The cell seeded decellularized vessel segments were implanted in thesubcutaneous space of athymic mice in order to test if HSVEC could bemaintained on the decllularized vessel in vivo. Vessel implants wereretrieved after 7 days and were processed for histological analysis. H&Estaining demonstrated a uniform monolayer of cells on the luminalsurface of the decellularized vessels. Immunohistochemical staining withanti-human CD31 confirmed that the cell layer consisted of HSVEC. Takentogether, these results indicate that the decellularized vessels are acompatible substrate for EC in vitro and in vivo.

Example 25 Characterization of Blood Vessels

This example described the various techniques used to characterize theseeded blood vessels:

(i) RNA Isolation and RT-PCR

Decellularized porcine vessels, vessels seeded with HSVEC (approximately4 cm) and, cultured HSVEC were homogenized in RNAzol reagent at 4° C.using a tissue homogenizer. RNA was isolated according to themanufacturer's protocol (TelTest, Friendswood, Tex.). Complementary DNAwas synthesized from 2 μg RNA using the Superscript II reversetranscriptase and random hexamers as primers. RT-PCR analysis usingFlt-1(5′ CTCAACAAGGATGCAGCACTACAC, 3′GGGAGCCATCCATTTCAGAGGAAGM 35cycles, annealing at 60° C.), KDR (5′ TTACAGATCTCCATTTATTGC,3′TTCATCTCACTCCCAGACT, 35 cycles annealing at 60° C.), neuropilin-1(NRP-1, 5′ TTTCGCAACGATAAATGTGGCGAT, 3′TATCACTCCACTAGGTGTTG, 30 cycles,annealing at 55° C.) and glyceraldehyde phosphate dehydrogenase (GAPDH)(5′ GTCTTCACCACCATGGAG, 3′CCACCCTGTTGCTGTAGC, 28 cycles annealing at 60°C.) primers (Invitrogen, Carlsbad, Calif.) was performed usingmanufacturers protocols. PCR products corresponding to Flt-1 (416 bp),KDR (479 bp), NRP-1 (409 bp) and GAPDH (673 bp) were resolved on a 2%agarose gel, stained with ethidium-bromide and visualized under U.V.light.

(ii) Synthesis of Prostaglandin F1α

Synthesis of Prostaglandin F1α was determined by measuring 6-ketoprostaglandin F1α in conditioned media. Briefly, HSVEC were seeded ontodecellularized matrices (approximately 1.5×10⁵ cells/50 mm²). After 48hours the matrices were transferred into separate wells of a 48-welldish. After 3 days, 50 μl of conditioned media were assayed using theenzyme immunoassay kit according to the manufacture's instructions(Cayman Chemical; Cayman Islands). In parallel, increasing amounts ofHSVEC were seeded on 48-well dishes and the media was changed when thecells completely adhered to the dish. Some well were counted in order todetermine the exact cell number. The amount of prostaglandin F_(1α) wasmeasured 24 hours later and multiplied by 3 in order to compare with theresults from the seeded vessels. Averages and standard deviations (SD)of 6-keto prostaglandin F_(1α) amounts were calculated and plottedagainst the number of cells.

(iii) Nitric Oxide (NO) Production

NO mediated vascular relaxation was evaluated in an organ-chamber.Briefly, guinea pig thoracic aorta was harvested, the endothelium layerwas removed by gentle rubbing and cut into 5-mm segments. Each segmentwas suspended between 2 tungsten stirrups for measurement of isometrictension. The vessel segments placed in an organ chamber with 10 mlKreb's buffer solution at 37° C. with a mixture of 5% CO₂, 15% O₂ and abalance of N₂. Each HSVEC seeded vessel (2-3 cm in length) was tied to a21 G needle, which was attached to plastic IV tubing and placed abovethe same organ chamber with the denuded aortic segment. The segmentswere contracted with 80 mM KCl Kreb's buffer in a stepwise fashion toobtain a resting tension of 4 g. After resting for 90 minutes, thesegments contracted in response to prostaglandin F2α up to a finalconcentration of 10⁻⁷ and until a stable contraction of approximately50% of maximum KCl-induced contraction was achieved. Inducers ofvasoactive agent secretion (A23187, a calcium ionophore) and antagonists(L-NAME) were then added using an infusion pump through the HSVEC seededvessels to induce NO production. Doses of the vasoactive agents between10⁻⁷ M-10⁻³ M were tested and dose-response curves were constructed.

To examine if HSVEC seeded on the decellularized vessels are functional,prostaglandin and nitric oxide metabolism in the cells was evaluated.The synthesis of 6-keto prostaglandin F1α, a potent inhibitor ofplatelet aggregation and a vascular smooth muscle relaxant was examined,as well as the hydrolysis product of PGI2 (FIG. 14A).

Decellularized vessel-seeded HSVEC secreted approximately 80 pg of6-keto prostaglandin (PG) F1α in 3 days. This level was similar to theamount of 6-keto PG F1a secreted by a sub-confluent monolayer (100,000cells) of HSVEC in a 75 mm² tissue culture dish. The ability of HSVEC toproduce NO was also examined using an organ-chamber methodology (FIG.14B). Endothelium-denuded segments of guinea-pig aortas were contractedwith Prostaglandin F2 alpha. The segments were exposed to increasingconcentrations of the calcium ionophore A23187 that was perfused throughHSVEC-seeded grafts in the same organ chamber. The guinea-pig aorticsegments relaxed in a dose-dependent manner. This dose-dependentrelaxation response to A23187 was significantly attenuated by adding1×10⁻³ M of the endothelial NO-synthase inhibitor L-NAME (75.0% versus10.7%, p<0.001). When A23187 was perfused through grafts not seeded withHSVEC, the guinea-pig aortic segments remained fully contracted.Finally, exposing the aortic segments to increasing concentrations ofsodium nitroprusside (SNP), an endothelial-independent NO donor,resulted in a dose-response relaxation, indicating normal smooth-musclefunction in the guinea-pig aortic segments. These results indicate thatHSVEC seeded on decellularized vessels are able to produce EC specificmetabolites in response to physiologic stimulus.

These results show that small diameter vascular grafts can be createdusing matrices such as decellularized matrix or nanospin matrices seededwith endothelial cells and using the bioreactor chamber and methods ofthe invention. Small-caliber blood vessels for clinical use by coatingdecellularized arteries with endothelial cells from a human source.Human saphenous vein endothelial cells (HSVEC) were successfullyisolated from discarded segments of saphenous vein and formed amonolayer on the decellularized vessels. These results suggest thathuman endothelial cells-seeded decellularized vessels can serve as aclinical alternative for small diameter blood vessels for surgicalpurposes.

The decellularized porcine arterial segments were the appropriatesize-range for vascular surgery, without the need to tubularizesheet-like matrices. The decellularized vessel matrix is composed oflayers of collagen surrounding a layer of elastin, as shown by Movatstaining. The multiple collagen layers contributed to the mechanicalstrength of the decellularized vessels that resisted pressures of 1,900mmHg without any noticeable leakage. The decellularization process didnot alter the porosity of the vessel wall as demonstrated by SEM. Thepresence of an elastin layer on the lumen of the vessels enhanced theadherence and growth of endothelial cells.

The presence of a confluent endothelial cells monolayer on small-caliberprosthetic grafts is essential to provide protection from thrombusformation following implantation. To avoid the immune response toxenografts, largely due to high immunogenicity of the non-humanendothelial cells, human endothelial cells were isolated from saphenousveins and used to seed the decellularized matrix. Saphenous vein-derivedendothelial cells were isolated by enzymatic digestion and reached thecritical amount of cells required for seeding a 5 cm long decellularizedvessel (approximately 5×10⁶ cells) after 10-14 days in-vitro. HSVECmaintained a typical endothelial cell monolayer for up to 12 passagesand expressed endothelial cells markers such as CD31, vWF and KDR(VEGFR-2).

The bioreactor chamber of the invention was used to efficiently seed thecells onto the decellularized vessels. The cells adhered to the luminalside of the vessel and formed a continuous monolayer that was preservedafter 7 days in vivo. These results indicate that the decellularizationprocess resulted in a biocompatible matrix composed of collagen andelastin and that the native matrix can support HSVEC growth.

It was further demonstrated that the seeded HSVEC synthesizedprostaglandin (PG) I2, as measured by levels of 6-keto PG F1a in theconditioned medium, and produced NO by an organ-chamber analysis. Thesepotent vasodilators, specifically secreted by endothelial cells inresponse to biochemical and mechanical stimuli are essential to maintainlong-term graft patency. The ability to synthesize vasoactive agentsfurther indicates that HSVEC can serve as a functional layer to coat thedecellularized vessel lumen. NO contributes to resting vascular tone,impairs platelet activation, and prevents leukocyte adhesion to theendothelium. These effects of NO on the vessel wall are important toprotect the implanted graft against early thrombosis and lateratherosclerosis.

Thus, the methods of the invention are useful for the generation offunctional blood vessels, particularly small-caliber blood vessels.

What is claimed is:
 1. A method for producing a preconditioned bloodvessel, comprising: providing a biocompatible matrix shaped in a tubularconfiguration seeded with a cultured population of endothelial cells onthe inside surface of the matrix, wherein the tubular matrix comprisesat least one nanoparticle coupled to a releasable therapeutic agentwhich is incorporated within the tubular matrix; attaching a first andsecond end of the tubular matrix to a first and second attachmentelement in a preconditioning chamber, wherein the first and secondattachment elements have a channel that is fluidly coupled to a fluidflow system; and preconditioning the seeded tubular matrix with the flowsystem by moving a biological fluid through the seeded tubular matrix,wherein the flow-rate and pulse-rate of the biological fluid iscontrolled such that the preconditioned blood vessel is produced.
 2. Themethod of claim 1, wherein the step of preconditioning the matrixcomprises moving the biological fluid through the inside surface of theseeded tubular matrix in a closed fluid flow system.
 3. The method ofclaim 1, wherein the step of providing a biocompatiable matrix furthercomprises: providing a biocompatible matrix shaped in a tubularconfiguration seeded with a cultured population of endothelial cells onthe inside surface of the matrix, wherein the tubular matrix comprisesat least one natural component from about 25 percent to about 75 percentby weight.
 4. The method of claim 1, further comprising: seeding thetubular matrix with a cultured population of endothelial cells on theinside surface of the matrix; and seeding the tubular matrix with apopulation of smooth muscle cells on the outside surface of the matrix.5. The method of claim 1, wherein the step of preconditioning the seededtubular matrix further comprises moving a biological fluid having acomposition and viscosity that mimics blood through the inside surfaceof the attached matrix as a pulsed flow to induce a wall shear stress ofat least 10 dynes/cm² so that the seeded matrix is exposed to fluid flowconditions that mimic flow of blood through a native blood vessel. 6.The method of claim 1, wherein the method further comprises continuingexposure of the cells on the inside surface of the matrix to the pulsedfluid flow to allow the seeded cells to develop under fluid flowconditions until the matrix can withstand a wall pressure distributionof at least 60 mmHg.
 7. The method of claim 1, wherein biocompatiblematrix is selected from the group consisting of a decellularized matrix,an electrospun matrix and a synthetic polymer matrix.
 8. The method ofclaim 1, wherein the preconditioning chamber is a container withdimensions suitable for attaching a length of tubular matrix.
 9. Themethod of claim 1, wherein the pulsed flow has a pulse-rate that isvaried over time to induce a wall shear stress in the range of about 10dynes/cm² to about 45 dynes/cm² .
 10. The method of claim 1, wherein thepulsed flow has a pulse-rate that is varied over time to induce a wallpressure distribution in the range of about 60 to about 200 mmHg. 11.The method of claim 1, wherein the biological fluid is moved through theseeded tubular matrix by a pump.
 12. The method of claim 1, wherein thebiological fluid is selected from the group consisting of culturemedium, buffer medium, and physiological medium.
 13. The method of claim1, further comprising adding a volume of biological fluid to thepreconditioning chamber such that the outside surface of the tubularmatrix is exposed to the biological fluid.
 14. The method of claim 1,wherein the tubular matrix is an electrospun matrix comprising at leastone natural component and at least one synthetic polymer component. 15.The method of claim 14, wherein the natural component is collagen andthe synthetic polymer component is poly(lactide-co-glycolides) (PLGA).16. The method of claim 1, wherein the matrix comprises elastin.
 17. Themethod of claim 1, further comprises applying radiation to heat thenanoparticle.
 18. The method of claim 17, wherein the step of applyingradiation comprises applying radiation at a wavelength in the range ofabout 700 nm to about 1000 nm to release the therapeutic agent from thenanoparticle.
 19. The method of claim 1, wherein the matrix furthercomprises an image enhancing agent.
 20. The method of claim 19, whereinthe image enhancing agent is gadolinium.
 21. The method of claim 1,wherein the endothelial cells are isolated from a human saphenous vein.22. The method of claim 1, wherein the endothelial cells are derivedfrom progenitor cells isolated from peripheral blood or bone marrow. 23.The method of claim 1, further comprising: evaluating production of atleast one vasoactive agent from the pre-conditioned seeded tubularmatrix, wherein production of at least one vasoactive agent indicatesthe preconditioned blood vessel is produced.